Methods of detecting cancer with antibodies to cytokine receptor ZCYTOR19

ABSTRACT

Novel polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods are disclosed for zcytor19, a novel class II cytokine receptor. The polypeptides may be used within methods for detecting ligands that stimulate the proliferation and/or development of hematopoietic, lymphoid and myeloid cells in vitro and in vivo. Ligand-binding receptor polypeptides can also be used to block ligand activity in vitro and in vivo. The polynucleotides encoding zcytor19, are located on chromosome 1p36.11, and can be used to identify a region of the genome associated with human disease states. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/165,141filed Jun. 23, 2005, which is a continuation of U.S. application Ser.No. 09/995,898, filed Nov. 28, 2001, now abandoned, both of which areherein incorporated by reference. U.S. application Ser. No. 09/995,898claims the benefit of U.S. Provisional Application 60/253,561, filed onNov. 28, 2000, and U.S. Provisional Application 60/267,211, filed onFeb. 7, 2001.

BACKGROUND OF THE INVENTION

Hormones and polypeptide growth factors control proliferation anddifferentiation of cells of multicellular organisms. These diffusablemolecules allow cells to communicate with each other and act in concertto form cells and organs, and to repair damaged tissue. Examples ofhormones and growth factors include the steroid hormones (e.g. estrogen,testosterone), parathyroid hormone, follicle stimulating hormone, theinterleukins, platelet derived growth factor (PDGF), epidermal growthfactor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF),erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding toreceptors. Receptors may be integral membrane proteins that are linkedto signaling pathways within the cell, such as second messenger systems.Other classes of receptors are soluble molecules, such as thetranscription factors. Of particular interest are receptors forcytokines, molecules that promote the proliferation and/ordifferentiation of cells. Examples of cytokines include erythropoietin(EPO), which stimulates the development of red blood cells;thrombopoietin (TPO), which stimulates development of cells of themegakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia, thrombocytopenia, and neutropenia or receiving chemotherapyfor cancer.

The demonstrated in vivo activities of these cytokines illustrate theenormous clinical potential of, and need for, other cytokines, cytokineagonists, and cytokine antagonists. The present invention addressesthese needs by providing new a hematopoietic cytokine receptor, as wellas related compositions and methods.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

DESCRIPTION OF THE INVENTION

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention.

Within one aspect, the present invention provides an isolatedpolynucleotide that encodes a polypeptide comprising an amino acidsequence selected from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) toamino acid number 223 (Pro); (b) the amino acid sequence as shown in SEQID NO:2 from amino acid number 21 (Arg) to amino acid number 226 (Asn);(c) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 21 (Arg) to amino acid number 249 (Trp); (d) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 250 (Lys) toamino acid number 491 (Arg); (e) the amino acid sequence as shown in SEQID NO:19 from amino acid number 250 (Lys) to 520 (Arg); (f) the aminoacid sequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) toamino acid number 491 (Arg); (g) the amino acid sequence as shown in SEQID NO:19 from amino acid number 21 (Arg) to amino acid number 520 (Arg);(h) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 1 (Met) to amino acid number 491 (Arg); (i) the amino acidsequence as shown in SEQ ID NO:19 from amino acid number 1 (Met) toamino acid number 520 (Arg); (j) the amino acid sequence as shown in SEQID NO:21 from amino acid number 21 (Arg) to amino acid number 163(Trp);(k) the amino acid sequence as shown in SEQ ID NO:21 from aminoacid number 21 (Arg) to amino acid number 211 (Ser); and (l) the aminoacid sequence as shown in SEQ ID NO:21 from amino acid number 1 (Met) toamino acid number 211 (Ser). In one embodiment, the isolatedpolynucleotide described above comprises a polynucleotide sequenceselected from the group consisting of: (a) a polynucleotide comprising anucleotide sequence as shown in SEQ ID NO:1 from nucleotide 61 tonucleotide 669; (b) a polynucleotide comprising a nucleotide sequence asshown in SEQ ID NO:1 from nucleotide 61 to 678; (c) a polynucleotidecomprising a nucleotide sequence as shown in SEQ ID NO:1 from nucleotide61 to nucleotide 747; (d) a polynucleotide comprising a nucleotidesequence as shown in SEQ ID NO:1 from nucleotide 748 to nucleotide 1473;(e) a polynucleotide comprising a nucleotide sequence as shown in SEQ IDNO:18 from nucleotide 748 to nucleotide 1560; (f) a polynucleotidecomprising a nucleotide sequence as shown in SEQ ID NO:1 from nucleotide61 to nucleotide 1473; (g) a polynucleotide comprising a nucleotidesequence as shown in SEQ ID NO:18 from nucleotide 61 to nucleotide 1560;(h) a polynucleotide comprising a nucleotide sequence as shown in SEQ IDNO:1 from nucleotide 1 to nucleotide 1473; (i) a polynucleotidecomprising a nucleotide sequence as shown in SEQ ID NO:18 fromnucleotide 1 to nucleotide 1560; (j) a polynucleotide comprising anucleotide sequence as shown in SEQ ID NO:20 from nucleotide 61 tonucleotide 489; (k) a polynucleotide comprising a nucleotide sequence asshown in SEQ ID NO:20 from nucleotide 61 to nucleotide 633; and (l) apolynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:20from nucleotide 1 to nucleotide 633. In another embodiment, the isolatedpolynucleotide is as described above, wherein the polynucleotide encodesa polypeptide that further comprises a transmembrane domain consistingof residues 227 (Trp) to 249 (Trp) of SEQ ID NO:2. In anotherembodiment, the isolated polynucleotide is as described above, whereinthe polynucleotide encodes a polypeptide that further comprises anintracellular domain consisting of residues 250 (Lys) to 491 (Arg) ofSEQ ID NO:2, or 250 (Lys) to 520 (Arg) of SEQ ID NO:19. In anotherembodiment, the isolated polynucleotide is as described above, whereinthe polypeptide encoded by the polynucleotide has activity as measuredby cell proliferation, activation of transcription of a reporter gene,or wherein the polypeptide encoded by the polynucleotide further bindsto an antibody, wherein the antibody is raised to a polypeptidecomprising a sequence of amino acids from the group consisting of: (a)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number21 (Arg) to amino acid number 223 (Pro); (b) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acidnumber 226 (Asn); (c) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 21 (Arg) to amino acid number 249 (Trp); (d) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 250(Lys) to amino acid number 491 (Arg); (e) the amino acid sequence asshown in SEQ ID NO:19 from amino acid number 250 (Lys) to 520 (Arg); (f)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number21 (Arg) to amino acid number 491 (Arg); (g) the amino acid sequence asshown in SEQ ID NO:19 from amino acid number 21 (Arg) to amino acidnumber 520 (Arg); (h) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 1 (Met) to amino acid number 491 (Arg); (i) theamino acid sequence as shown in SEQ ID NO:19 from amino acid number 1(Met) to amino acid number 520 (Arg); (j) the amino acid sequence asshown in SEQ ID NO:21 from amino acid number 21 (Arg) to amino acidnumber 163 (Trp); (k) the amino acid sequence as shown in SEQ ID NO:21from amino acid number 21 (Arg) to amino acid number 211 (Ser); and (l)the amino acid sequence as shown in SEQ ID NO:21 from amino acid number1 (Met) to amino acid number 211 (Ser); and wherein the binding of theantibody to the isolated polypeptide is measured by a biological orbiochemical assay including radioimmunoassay, radioimmuno-precipitation,Western blot, or enzyme-linked immunosorbent assay.

Within a second aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide from thegroup consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 21 (Arg) to amino acid number 223 (Pro); (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 21(Arg) to amino acid number 226 (Asn); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acidnumber 249 (Trp); (d) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 250 (Lys) to amino acid number 491 (Arg); (e) theamino acid sequence as shown in SEQ ID NO:19 from amino acid number 250(Lys) to 520 (Arg); (f) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 21 (Arg) to amino acid number 491 (Arg); (g) theamino acid sequence as shown in SEQ ID NO:19 from amino acid number 21(Arg) to amino acid number 520 (Arg); (h) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number491 (Arg); (i) the amino acid sequence as shown in SEQ ID NO:19 fromamino acid number 1 (Met) to amino acid number 520 (Arg); (j) the aminoacid sequence as shown in SEQ ID NO:21 from amino acid number 21 (Arg)to amino acid number 163 (Trp); (k) the amino acid sequence as shown inSEQ ID NO:21 from amino acid number 21 (Arg) to amino acid number 211(Ser); and (l) the amino acid sequence as shown in SEQ ID NO:21 fromamino acid number 1 (Met) to amino acid number 211 (Ser); and atranscription terminator, wherein the promoter is operably linked to theDNA segment, and the DNA segment is operably linked to the transcriptionterminator. In one embodiment, the expression vector described abovefurther comprises a secretory signal sequence operably linked to the DNAsegment.

Within a third aspect, the present invention provides a cultured cellcomprising an expression vector according to claim 7, wherein the cellexpresses a polypeptide encoded by the DNA segment. In anotherembodiment, the expression vector is as described above, wherein thepolypeptide further comprises a transmembrane domain consisting ofresidues 227 (Trp) to 249 (Trp) of SEQ ID NO:2. In another embodiment,the expression vector is as described above, wherein the polypeptidefurther comprises an intracellular domain consisting of residues 250(Lys) to 491 (Arg) of SEQ ID NO:2 or 250 (Lys) to 520 (Arg) of SEQ IDNO:19. In another embodiment, the expression vector is as describedabove, comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide from thegroup consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 21 (Arg) to amino acid number 223 (Pro); (b) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 21(Arg) to amino acid number 226 (Asn); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acidnumber 249 (Trp); (d) the amino acid sequence as shown in SEQ ID NO:21from amino acid number 21 (Arg) to amino acid number 211 (Ser); and atranscription terminator, wherein the promoter is operably linked to theDNA segment, and the DNA segment is operably linked to the transcriptionterminator.

Within another aspect, the present invention provides a cultured cellcomprising an expression vector according to claim 7, wherein the cellexpresses a polypeptide encoded by the DNA segment cultured cell intowhich has been introduced an expression vector according to claim 11,wherein the cell expresses a soluble receptor polypeptide encoded by theDNA segment.

Within another aspect, the present invention provides a DNA constructencoding a fusion protein, the DNA construct comprising: a first DNAsegment encoding a polypeptide comprising a sequence of amino acidresidues selected from the group consisting of: (a) the amino acidsequence of SEQ ID NO:2 from amino acid number 1 (Met), to amino acidnumber 20 (Gly); (b) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 21 (Arg) to amino acid number 223 (Pro); (c) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 21(Arg) to amino acid number 226 (Asn); (d) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acidnumber 249 (Trp); (e) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 250 (Lys) to amino acid number 491 (Arg); (f) theamino acid sequence as shown in SEQ ID NO:19 from amino acid number 250(Lys) to amino acid number 520 (Arg); (g) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 227 (Trp) to amino acidnumber 249 (Trp); (h) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 227 (Trp) to amino acid number 491 (Arg); (i) theamino acid sequence as shown in SEQ ID NO:19 from amino acid number 227(Trp) to amino acid number 520 (Arg); (j) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acidnumber 491 (Arg); (k) the amino acid sequence as shown in SEQ ID NO:19from amino acid number 21 (Arg) to amino acid number 520 (Arg); (l) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 1(Met) to amino acid number 491 (Arg); and (m) the amino acid sequence asshown in SEQ ID NO:19 from amino acid number 1 (Met) to amino acidnumber 520 (Arg); (n) the amino acid sequence as shown in SEQ ID NO:21from amino acid number 21 (Arg) to amino acid number 163 (Trp); (o) theamino acid sequence as shown in SEQ ID NO:21 from amino acid number 21(Arg) to amino acid number 211 (Ser); and (p) the amino acid sequence asshown in SEQ ID NO:21 from amino acid number 1 (Met) to amino acidnumber 211 (Ser); and at least one other DNA segment encoding anadditional polypeptide, wherein the first and other DNA segments areconnected in-frame; and wherein the first and other DNA segments encodethe fusion protein.

Within another aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA construct encoding a fusion proteinaccording to claim 13; and a transcription terminator, wherein thepromoter is operably linked to the DNA construct, and the DNA constructis operably linked to the transcription terminator.

Within another aspect, the present invention provides a cultured cellcomprising an expression vector according to claim 14, wherein the cellexpresses a polypeptide encoded by the DNA construct.

Within another aspect, the present invention provides a method ofproducing a fusion protein comprising: culturing a cell according toclaim 15; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides an isolatedpolypeptide comprising a sequence of amino acid residues selected fromthe group consisting of: (a) the amino acid sequence as shown in SEQ IDNO:2 from amino acid number 21 (Arg) to amino acid number 223 (Pro); (b)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number21 (Arg) to amino acid number 226 (Asn); (c) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acidnumber 249 (Trp); (d) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 250 (Lys) to amino acid number 491 (Arg); (e) theamino acid sequence as shown in SEQ ID NO:19 from amino acid number 250(Lys) to 520 (Arg); (f) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 21 (Arg) to amino acid number 491 (Arg); (g) theamino acid sequence as shown in SEQ ID NO:19 from amino acid number 21(Arg) to amino acid number 520 (Arg); (h) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number491 (Arg); (i) the amino acid sequence as shown in SEQ ID NO:19 fromamino acid number 1 (Met) to amino acid number 520 (Arg); (j) the aminoacid sequence as shown in SEQ ID NO:21 from amino acid number 21 (Arg)to amino acid number 163 (Trp); (k) the amino acid sequence as shown inSEQ ID NO:21 from amino acid number 21 (Arg) to amino acid number 211(Ser); and (l) the amino acid sequence as shown in SEQ ID NO:21 fromamino acid number 1 (Met) to amino acid number 211 (Ser). In oneembodiment, the isolated polypeptide is as described above, wherein thepolypeptide consists of a sequence of amino acid residues that isselected from the group consisting of: (a) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acidnumber 223 (Pro); (b) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 21 (Arg) to amino acid number 226 (Asn); (c) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 21(Arg) to amino acid number 249 (Trp); (d) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 250 (Lys) to amino acidnumber 491 (Arg); (e) the amino acid sequence as shown in SEQ ID NO:19from amino acid number 250 (Lys) to 520 (Arg); (f) the amino acidsequence as shown in SEQ ID NO:2 from amino acid number 21 (Arg) toamino acid number 491 (Arg); (g) the amino acid sequence as shown in SEQID NO:19 from amino acid number 21 (Arg) to amino acid number 520 (Arg);(h) the amino acid sequence as shown in SEQ ID NO:2 from amino acidnumber 1 (Met) to amino acid number 491 (Arg); (i) the amino acidsequence as shown in SEQ ID NO:19 from amino acid number 1 (Met) toamino acid number 520 (Arg); (j) the amino acid sequence as shown in SEQID NO:21 from amino acid number 21 (Arg) to amino acid number 163 (Trp);(k) the amino acid sequence as shown in SEQ ID NO:21 from amino acidnumber 21 (Arg) to amino acid number 211 (Ser); and (l) the amino acidsequence as shown in SEQ ID NO:21 from amino acid number 1 (Met) toamino acid number 211 (Ser). In another embodiment, the isolatedpolypeptide is as described above, wherein the polypeptide furthercomprises a transmembrane domain consisting of residues 227 (Trp) to 249(Trp) of SEQ ID NO:2. In another embodiment, the isolated polypeptide isas described above, wherein the polypeptide further comprises anintracellular domain consisting of residues 250 (Lys) to amino acidnumber 491 (Arg) of SEQ ID NO:2, or 250 (Lys) to amino acid number 520(Arg) of SEQ ID NO:19. In another embodiment, the isolated polypeptideis as described above, wherein the polypeptide has activity as measuredby cell proliferation, activation of transcription of a reporter gene,or wherein the polypeptide encoded by the polynucleotide further bindsto an antibody, wherein the antibody is raised to a polypeptidecomprising a sequence of amino acids from the group consisting of: (a)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number21 (Arg) to amino acid number 223 (Pro); (b) the amino acid sequence asshown in SEQ ID NO:2 from amino acid number 21 (Arg) to amino acidnumber 226 (Asn); (c) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 21 (Arg) to amino acid number 249 (Trp); (d) theamino acid sequence as shown in SEQ ID NO:2 from amino acid number 250(Lys) to amino acid number 491 (Arg); (e) the amino acid sequence asshown in SEQ ID NO:19 from amino acid number 250 (Lys) to 520 (Arg); (f)the amino acid sequence as shown in SEQ ID NO:2 from amino acid number21 (Arg) to amino acid number 491 (Arg); (g) the amino acid sequence asshown in SEQ ID NO:19 from amino acid number 21 (Arg) to amino acidnumber 520 (Arg); (h) the amino acid sequence as shown in SEQ ID NO:2from amino acid number 1 (Met) to amino acid number 491 (Arg); (i) theamino acid sequence as shown in SEQ ID NO:19 from amino acid number 1(Met) to amino acid number 520 (Arg); (j) the amino acid sequence asshown in SEQ ID NO:21 from amino acid number 21 (Arg) to amino acidnumber 163 (Trp); (k) the amino acid sequence as shown in SEQ ID NO:21from amino acid number 21 (Arg) to amino acid number 211 (Ser); and (l)the amino acid sequence as shown in SEQ ID NO:21 from amino acid number1 (Met) to amino acid number 211 (Ser); and wherein the binding of theantibody to the isolated polypeptide is measured by a biological orbiochemical assay including radioimmunoassay, radioimmuno-precipitation,Western blot, or enzyme-linked immunosorbent assay.

Within another aspect, the present invention provides a method ofproducing a polypeptide comprising: culturing a cell according to claim8; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides an isolatedpolypeptide comprising an amino acid segment selected from the groupconsisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 fromamino acid number 21 (Arg) to amino acid number 226 (Asn)); (b) theamino acid sequence as shown in SEQ ID NO:4; (c) the amino acid sequenceas shown in SEQ ID NO:21 from amino acid number 21 (Arg) to amino acidnumber 211 (Ser); and (c) sequences that are at least 90% identical to(a), (b) or (c), wherein the polypeptide is substantially free oftransmembrane and intracellular domains ordinarily associated withhematopoietic receptors.

Within another aspect, the present invention provides a method ofproducing a polypeptide comprising: culturing a cell according to claim12; and isolating the polypeptide produced by the cell.

Within another aspect, the present invention provides a method ofproducing an antibody to a polypeptide comprising: inoculating an animalwith a polypeptide selected from the group consisting of: (a) apolypeptide consisting of 50 to 471 amino acids, wherein the polypeptidecomprises a contiguous sequence of amino acids in SEQ ID NO:2 from aminoacid number 21 (Arg), to amino acid number 491 (Arg); (b) a polypeptideconsisting of 50 to 500 amino acids, wherein the polypeptide comprises acontiguous sequence of amino acids in SEQ ID NO:19 from amino acidnumber 21 (Arg), to amino acid number 520 (Arg); (c) a polypeptideconsisting of 50 to 191 amino acids, wherein the polypeptide comprises acontiguous sequence of amino acids in SEQ ID NO:21 from amino acidnumber 21 (Arg), to amino acid number 211 (Ser); (d) a polypeptideaccording to claim 18; (e) a polypeptide comprising amino acid number 21(Arg) to 119 (Tyr) of SEQ ID NO:2; (f) a polypeptide comprising aminoacid number 125 (Pro) to 223 (Pro) of SEQ ID NO:2; (g) a polypeptidecomprising a hydrophilic peptide of SEQ ID NO:2 as predicted from ahydrophobicity plot using a Hopp/Woods hydrophilicity profile based on asliding six-residue window, with buried G, S, and T residues and exposedH, Y, and W residues ignored; and wherein the polypeptide elicits animmune response in the animal to produce the antibody; and isolating theantibody from the animal.

Within another aspect, the present invention provides an antibodyproduced by the method of claim 25, which specifically binds to apolypeptide of SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21. In oneembodiment, the antibody described above is a monoclonal antibody.

Within another aspect, the present invention provides an antibody thatspecifically binds to a polypeptide as disclosed above.

Within another aspect, the present invention provides a method ofdetecting, in a test sample, the presence of a modulator of the activityof a cytokine receptor protein comprising: culturing a cell into whichhas been introduced an expression vector according to claim 6, whereinthe cell expresses the protein encoded by the DNA segment in thepresence and absence of a test sample; and comparing levels of activityof the protein in the presence and absence of a test sample, by abiological or biochemical assay; and determining from the comparison,the presence of modulator the cytokine receptor protein activity in thetest sample.

Within another aspect, the present invention provides a method fordetecting a cytokine receptor ligand within a test sample, comprising:contacting a test sample with a cytokine receptor polypeptide comprisingan amino acid sequence from the group consisting of: (a) the amino acidsequence as shown in SEQ ID NO:4; (b) the amino acid sequence as shownin SEQ ID NO:2 from amino acid number 21 (Arg) to amino acid number 226(Asn); and (c) the amino acid sequence as shown in SEQ ID NO:21 fromamino acid number 21 (Arg) to amino acid number 211 (Ser); and detectingthe binding of the cytokine receptor polypeptide to a ligand in thesample. In one embodiment, the method for detecting a cytokine receptorligand is as disclosed above, wherein the cytokine receptor polypeptideis membrane bound within a cultured cell, and the detecting stepcomprises measuring a biological response in the cultured cell. Inanother embodiment, the method for detecting a cytokine receptor ligandis as disclosed above, wherein the biological response is cellproliferation or activation of transcription of a reporter gene.

Within another aspect, the present invention provides a method fordetecting a genetic abnormality in a patient, comprising: obtaining agenetic sample from a patient; producing a first reaction product byincubating the genetic sample with a polynucleotide comprising at least14 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:18 or SEQ ID NO:20or the complement of SEQ ID NO:1, SEQ ID NO:18 or SEQ ID NO:20, underconditions wherein said polynucleotide will hybridize to complementarypolynucleotide sequence; visualizing the first reaction product; andcomparing said first reaction product to a control reaction product froma wild type patient, wherein a difference between said first reactionproduct and said control reaction product is indicative of a geneticabnormality in the patient.

Within another aspect, the present invention provides a method fordetecting a cancer in a patient, comprising: obtaining a tissue orbiological sample from a patient; incubating the tissue or biologicalsample with an antibody of claim 29 under conditions wherein theantibody binds to its complementary polypeptide in the tissue orbiological sample; visualizing the antibody bound in the tissue orbiological sample; and comparing levels of antibody bound in the tissueor biological sample from the patient to a normal control tissue orbiological sample, wherein an increase in the level of antibody bound tothe patient tissue or biological sample relative to the normal controltissue or biological sample is indicative of a cancer in the patient.

Within another aspect, the present invention provides a method fordetecting a cancer in a patient, comprising:

obtaining a tissue or biological sample from a patient;

labeling a polynucleotide comprising at least 14 contiguous nucleotidesof SEQ ID NO:1, SEQ ID NO:18 or SEQ ID NO:20 or the complement of SEQ IDNO:1, SEQ ID NO:18 or SEQ ID NO:20;

incubating the tissue or biological sample with under conditions whereinthe polynucleotide will hybridize to complementary polynucleotidesequence;

visualizing the labeled polynucleotide in the tissue or biologicalsample; and

comparing the level of labeled polynucleotide hybridization in thetissue or biological sample from the patient to a normal control tissueor biological sample,

wherein an increase in the labeled polynucleotide hybridization to thepatient tissue or biological sample relative to the normal controltissue or biological sample is indicative of a cancer in the patient.

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” is a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence. For example, the sequence 5′ ATGCACGGG3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers, multimers, or alternativelyglycosylated or derivatized forms.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

“Probes and/or primers” as used herein can be RNA or DNA. DNA can beeither cDNA or genomic DNA. Polynucleotide probes and primers are singleor double-stranded DNA or RNA, generally synthetic oligonucleotides, butmay be generated from cloned cDNA or genomic sequences or itscomplements. Analytical probes will generally be at least 20 nucleotidesin length, although somewhat shorter probes (14-17 nucleotides) can beused. PCR primers are at least 5 nucleotides in length, preferably 15 ormore nt, more preferably 20-30 nt. Short polynucleotides can be usedwhen a small region of the gene is targeted for analysis. For grossanalysis of genes, a polynucleotide probe may comprise an entire exon ormore. Probes can be labeled to provide a detectable signal, such as withan enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,paramagnetic particle and the like, which are commercially availablefrom many sources, such as Molecular Probes, Inc., Eugene, Oreg., andAmersham Corp., Arlington Heights, Ill., using techniques that are wellknown in the art.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” is used herein to denote a cell-associated protein,or a polypeptide subunit of such a protein, that binds to a bioactivemolecule (the “ligand”) and mediates the effect of the ligand on thecell. Binding of ligand to receptor results in a conformational changein the receptor (and, in some cases, receptor multimerization, i.e.,association of identical or different receptor subunits) that causesinteractions between the effector domain(s) and other molecule(s) in thecell. These interactions in turn lead to alterations in the metabolismof the cell. Metabolic events that are linked to receptor-ligandinteractions include gene transcription, phosphorylation,dephosphorylation, cell proliferation, increases in cyclic AMPproduction, mobilization of cellular calcium, mobilization of membranelipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis ofphospholipids. Cell-surface cytokine receptors are characterized by amulti-domain structure as discussed in more detail below. Thesereceptors are anchored in the cell membrane by a transmembrane domaincharacterized by a sequence of hydrophobic amino acid residues(typically about 21-25 residues), which is commonly flanked bypositively charged residues (Lys or Arg). In general, receptors can bemembrane bound, cytosolic or nuclear; monomeric (e.g., thyroidstimulating hormone receptor, beta-adrenergic receptor) or multimeric(e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSFreceptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).The term “receptor polypeptide” is used to denote complete receptorpolypeptide chains and portions thereof, including isolated functionaldomains (e.g., ligand-binding domains). The terms “ligand-bindingdomain(s)” and “cytokine-binding domain(s)” can be used interchangeably.

A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

Cytokine receptor subunits are characterized by a multi-domain structurecomprising a ligand-binding domain and an effector domain that istypically involved in signal transduction. Multimeric cytokine receptorsinclude homodimers (e.g., PDGF receptor αα and ββ isoforms,erythropoietin receptor, MPL (thrombopoietin receptor), and G-CSFreceptor); heterodimers whose subunits each have ligand-binding andeffector domains (e.g., PDGF receptor αβ isoform); and multimers havingcomponent subunits with disparate functions (e.g., IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor subunits arecommon to a plurality of receptors. For example, the AIC2B subunit,which cannot bind ligand on its own but includes an intracellular signaltransduction domain, is a component of IL-3 and GM-CSF receptors. Manycytokine receptors can be placed into one of four related families onthe basis of their structures and functions. Class I hematopoieticreceptors, for example, are characterized by the presence of a domaincontaining conserved cysteine residues and the WSXWS motif (SEQ IDNO:5). Additional domains, including protein kinase domains; fibronectintype III domains; and immunoglobulin domains, which are characterized bydisulfide-bonded loops, are present in certain hematopoietic receptors.Cytokine receptor structure has been reviewed by Urdal, Ann. ReportsMed. Chem. 26:221-228, 1991 and Cosman, Cytokine 5:95-106, 1993. It isgenerally believed that under selective pressure for organisms toacquire new biological functions, new receptor family members arose fromduplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members.

Cell-surface cytokine receptors are further characterized by thepresence of additional domains. These receptors are anchored in the cellmembrane by a transmembrane domain characterized by a sequence ofhydrophobic amino acid residues (typically about 21-25 residues), whichis commonly flanked by positively charged residues (Lys or Arg). On theopposite end of the protein from the extracellular domain and separatedfrom it by the transmembrane domain is an intracellular domain.

The Zcytor19 receptor of the present invention is a class II cytokinereceptor. These receptors usually bind to four-helix-bundle cytokines.Interleukin-10 and the interferons have receptors in this class (e.g.,interferon-gamma, alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains). Class II cytokine receptors arecharacterized by the presence of one or more cytokine receptor modules(CRM) in their extracellular domains. Other class II cytokine receptorsinclude zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4(Genbank Accession No. Z17227), IL-10R (Genbank Accession No.s U00672and NM_(—)001558), DIRS1, zcytor7 (commonly owned U.S. Pat. No.5,945,511), zcytor16, tissue factor, and the like. The CRMs of class IIcytokine receptors are somewhat different than the better-known CRMs ofclass I cytokine receptors. While the class II CRMs contain two type-IIIfibronectin-like domains, they differ in organization.

Zcytor19, like all known class II receptors except interferon-alpha/betareceptor alpha chain, has only a single class II CRM in itsextracellular domain. Zcytor19 is a receptor for a helical cytokine ofthe interferon/IL-10 class. As was stated above, Zcytor19 is similar toother Class II cytokine receptors such as zcytor11 and zcytor16.Analysis of a human cDNA clone encoding Zcytor19 (SEQ ID NO:1) revealedan open reading frame encoding 491 amino acids (SEQ ID NO:2) comprisinga secretory signal sequence (residues 1 (Met) to 20 (Gly) of SEQ IDNO:2) and a mature zcytor19 cytokine receptor polyptide (residues 21(Arg) to 491 (Arg) of SEQ ID NO:2) an extracellular ligand-bindingdomain of approximately 206 amino acid residues (residues 21 (Arg) to226 (Asn) of SEQ ID NO:2), a transmembrane domain of approximately 23amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID NO:2),and an intracellular domain of approximately 242 amino acid residues(residues 250 (Lys) to 491 (Arg) of SEQ ID NO:2). Within theextracellular ligand-binding domain, there are two fibronectin type IIIdomains and a linker region. The first fibronectin type III domaincomprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:2, the linkercomprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:2, and the secondfibronectin type III domain is short, and comprises residues 125 (Pro)to 223 (Pro) of SEQ ID NO:2. Thus, a polypeptide comprising amino acids21 (Arg) to 223 (Pro) of SEQ ID NO:2 (SEQ ID NO:4) is considered aligand binding fragment. In addition as typically conserved in class IIreceptors, there are conserved Tryptophan residues comprising residues43 (Trp) and 68 (Trp) as shown in SEQ ID NO:2, and conserved Cysteineresidues at positions 74, 82, 195, 217 of SEQ ID NO:2.

In addition, the present invention includes a variant of zcytor19receptor that includes an approximately 30 amino acid insertion in theintracellular domain of the polyeptide (in reference to SEQ ID NO:2).Analysis of a human cDNA clone encoding Zcytor19 (SEQ ID NO:18) revealedan open reading frame encoding 520 amino acids (SEQ ID NO:19) comprisinga secretory signal sequence (residues 1 (Met) to 20 (Gly) of SEQ IDNO:19) and a mature zcytor19 cytokine receptor polypeptide (residues 21(Arg) to 520 (Arg) of SEQ ID NO:19) an extracellular ligand-bindingdomain of approximately 206 amino acid residues (residues 21 (Arg) to226 (Asn) of SEQ ID NO:19), a transmembrane domain of approximately 23amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID NO:19),and an intracellular domain of approximately 271 amino acid residues(residues 250 (Lys) to 520 (Arg) of SEQ ID NO:19). Within theextracellular ligand-binding domain, there are two fibronectin type IIIdomains and a linker region. The first fibronectin type III domaincomprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:19, the linkercomprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:19, and thesecond fibronectin type III domain comprises residues 125 (Pro) to 223(Pro) of SEQ ID NO:19. Thus, a polypeptide comprising amino acids 21(Arg) to 223 (Pro) of SEQ ID NO:19 (SEQ ID NO:4) is considered a ligandbinding fragment. In addition as typically conserved in class IIreceptors, there are conserved Tryptophan residues comprising residues43 (Trp) and 68 (Trp) as shown in SEQ ID NO:19, and conserved Cysteineresidues at positions 74, 82, 195, 217 of SEQ ID NO:19.

Moreover, a truncated soluble form of the zcytor19 receptor polypeptideappears to be naturally expressed. Analysis of a human cDNA cloneencoding the truncated soluble Zcytor19 (SEQ ID NO:20) revealed an openreading frame encoding 211 amino acids (SEQ ID NO:21) comprising asecretory signal sequence (residues 1 (Met) to 20 (Gly) of SEQ ID NO:21)and a mature truncated soluble zcytor19 receptor polyptide (residues 21(Arg) to 211 (Ser) of SEQ ID NO:21) a truncated extracellularligand-binding domain of approximately 143 amino acid residues (residues21 (Arg) to 163 (Trp) of SEQ ID NO:21), no transmembrane domain, but anadditional domain of approximately 48 amino acid residues (residues 164(Lys) to 211 (Ser) of SEQ ID NO:21). Within the truncated extracellularligand-binding domain, there are two fibronectin type III domains and alinker region. The first fibronectin type III domain comprises residues21 (Arg) to 119 (Tyr) of SEQ ID NO:21, the linker comprises residues 120(Leu) to 124 (Glu) of SEQ ID NO:21, and the second fibronectin type IIIdomain comprises residues 125 (Pro) to 163 (Trp) of SEQ ID NO:21. Thus,a polypeptide comprising amino acids 21 (Arg) to 163 (Trp) of SEQ IDNO:21 is considered a ligand binding fragment. In addition as typicallyconserved in class II receptors, there are conserved Tryptophan residuescomprising residues 43 (Trp) and 68 (Trp) as shown in SEQ ID NO:21, andconserved Cysteine residues in this truncated soluble form of thezcytor19 receptor are at positions 74, and 82 of SEQ ID NO:21.

Moreover, the zcytor19 polypeptide of the present invention can benaturally expressed wherein the extracellular ligand binding domaincomprises an additional 5-15 amino acid residues at the N-terminus ofthe mature polypeptide, or extracellular cytokine binding domain orcytokine binding fragment, as described above.

Those skilled in the art will recognize that these domain boundaries areapproximate and are based on alignments with known proteins andpredictions of protein folding. Deletion of residues from the ends ofthe domains is possible. Moreover the regions, domains and motifsdescribed above in reference to SEQ ID NO:2 are also as shown in SEQ IDNO:1; domains and motifs described above in reference to SEQ ID NO:19are also as shown in SEQ ID NO:18; and domains and motifs describedabove in reference to SEQ ID NO:21 are also as shown in SEQ ID NO:20.

The presence of transmembrane regions, and conserved and low variancemotifs generally correlates with or defines important structural regionsin proteins. Regions of low variance (e.g., hydrophobic clusters) aregenerally present in regions of structural importance (Sheppard, P. etal., Gene 150:163-167, 1994). Such regions of low variance often containrare or infrequent amino acids, such as Tryptophan. The regions flankingand between such conserved and low variance motifs may be more variable,but are often functionally significant because they may relate to ordefine important structures and activities such as binding domains,biological and enzymatic activity, signal transduction, cell-cellinteraction, tissue localization domains and the like.

The regions of conserved amino acid residues in zcytor19, describedabove, can be used as tools to identify new family members. Forinstance, reverse transcription-polymerase chain reaction (RT-PCR) canbe used to amplify sequences encoding the conserved regions from RNAobtained from a variety of tissue sources or cell lines. In particular,highly degenerate primers designed from the zcytor19 sequences areuseful for this purpose. Designing and using such degenerate primers maybe readily performed by one of skill in the art.

The present invention provides polynucleotide molecules, including DNAand RNA molecules that encode the zcytor19 polypeptides disclosedherein. Those skilled in the art will recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:3 is adegenerate DNA sequence that encompass all DNAs that encode the zcytor19polypeptide of SEQ ID NO:2; SEQ ID NO:28 is a degenerate DNA sequencethat encompass all DNAs that encode the zcytor19 polypeptide of SEQ IDNO:19; and SEQ ID NO:29 is a degenerate DNA sequence that encompass allDNAs that encode the zcytor19 polypeptide of SEQ ID NO:21. Those skilledin the art will recognize that the degenerate sequences of SEQ ID NO:3,SEQ ID NO:28, and SEQ ID NO:29 also provide all RNA sequences encodingSEQ ID NO:2, SEQ ID NO:19, and SEQ ID NO:21 by substituting U for T.Thus, zcytor19 polypeptide-encoding polynucleotides comprisingnucleotide 1 to nucleotide 1473 of SEQ ID NO:3, 1 to nucleotide 1560 ofSEQ ID NO:28, 1 to nucleotide 633 of SEQ ID NO:29, and their RNAequivalents are contemplated by the present invention. Moreover,subfragments of these degenerate sequences such as the mature forms ofthe polypeptides, extracellular, cytokine binding domains, intracellulardomains, and the like, as described herein are included in the presentinvention. One of skill in the art upon reference to SEQ ID NO:2, SEQ IDNO:19 and SEQ ID NO:21 and the subfragments thereof described hereincould readily determine the respective nucleotides in SEQ ID NO:3, SEQID NO:28 or SEQ ID NO:29, that encode those subfragments. Table 1 setsforth the one-letter codes used within SEQ ID NO:3, to denote degeneratenucleotide positions. “Resolutions” are the nucleotides denoted by acode letter. “Complement” indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C or T, and itscomplement R denotes A or G, A being complementary to T, and G beingcomplementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:3, encompassing all possiblecodons for a given amino acid, are set forth in Table 2.

TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:19, and/or SEQ ID NO:21. Variantsequences can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:3 serves as templates for optimizing expression of zcytor19polynucleotides in various cell types and species commonly used in theart and disclosed herein. Sequences containing preferential codons canbe tested and optimized for expression in various species, and testedfor functionality as disclosed herein.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,SEQ ID NO:18, or SEQ ID NO:20, or a sequence complementary thereto,under stringent conditions. In general, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences (e.g., >50 base pairs) is performed at temperatures of about20-25° C. below the calculated T_(m). For smaller probes (e.g., <50 basepairs) hybridization is typically carried out at the T_(m) or 5-10° C.below. This allows for the maximum rate of hybridization for DNA-DNA andDNA-RNA hybrids. Higher degrees of stringency at lower temperatures canbe achieved with the addition of formamide which reduces the T_(m) ofthe hybrid about 1° C. for each 1% formamide in the buffer solution.Suitable stringent hybridization conditions are equivalent to about a 5h to overnight incubation at about 42° C. in a solution comprising:about 40-50% formamide, up to about 6×SSC, about 5× Denhardt's solution,zero up to about 10% dextran sulfate, and about 10-20 μg/ml denaturedcommercially-available carrier DNA. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide; hybridization is then followed bywashing filters in up to about 2×SSC. For example, a suitable washstringency is equivalent to 0.1×SSC to 2×SSC, 0.1% SDS, at 55° C. to 65°C. Different degrees of stringency can be used during hybridization andwashing to achieve maximum specific binding to the target sequence.Typically, the washes following hybridization are performed atincreasing degrees of stringency to remove non-hybridized polynucleotideprobes from hybridized complexes. Stringent hybridization and washconditions depend on the length of the probe, reflected in the Tm,hybridization and wash solutions used, and are routinely determinedempirically by one of skill in the art.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zcytor19 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include PBLs, spleen, thymus, bone marrow, and lymphtissues, human erythroleukemia cell lines, acute monocytic leukemia celllines, B-cell and T-cell leukemia tissue or cell lines, other lymphoidand hematopoietic cell lines, and the like. Total RNA can be preparedusing guanidinium isothiocyanate extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using knownmethods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding zcytor19 polypeptides are then identified andisolated by, for example, hybridization or polymerase chain reaction(PCR) (Mullis, U.S. Pat. No. 4,683,202).

A full-length clone encoding zcytor19 can be obtained by conventionalcloning procedures. Complementary DNA (cDNA) clones are preferred,although for some applications (e.g., expression in transgenic animals)it may be preferable to use a genomic clone, or to modify a cDNA cloneto include at least one genomic intron. Methods for preparing cDNA andgenomic clones are well known and within the level of ordinary skill inthe art, and include the use of the sequence disclosed herein, or partsthereof, for probing or priming a library. Expression libraries can beprobed with antibodies to zcytor19, receptor fragments, or otherspecific binding partners.

The polynucleotides of the present invention can also be synthesizedusing DNA synthesis machines. Currently the method of choice is thephosphoramidite method. If chemically synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short polynucleotides (60 to 80 bp) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. However, for producinglonger polynucleotides (>300 bp), special strategies are usuallyemployed, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length.

An alternative way to prepare a full-length gene is to synthesize aspecified set of overlapping oligonucleotides (40 to 100 nucleotides).After the 3′ and 5′ short overlapping complementary regions (6 to 10nucleotides) are annealed, large gaps still remain, but the shortbase-paired regions are both long enough and stable enough to hold thestructure together. The gaps are filled and the DNA duplex is completedvia enzymatic DNA synthesis by E. coli DNA polymerase I. After theenzymatic synthesis is completed, the nicks are sealed with T4 DNAligase. Double-stranded constructs are sequentially linked to oneanother to form the entire gene sequence which is verified by DNAsequence analysis. See Glick and Pasternak, Molecular Biotechnology,Principles & Applications of Recombinant DNA, (ASM Press, Washington,D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 andClimie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990. Moreover,other sequences are generally added that contain signals for properinitiation and termination of transcription and translation.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are zcytor19 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human zcytor19 can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses zcytor19 as disclosed herein. Suitable sources of mRNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from mRNA of apositive tissue or cell line. A zcytor19-encoding cDNA can then beisolated by a variety of methods, such as by probing with a complete orpartial human cDNA or with one or more sets of degenerate probes basedon the disclosed sequences. A cDNA can also be cloned using PCR (Mullis,supra.), using primers designed from the representative human zcytor19sequence disclosed herein. Within an additional method, the cDNA librarycan be used to transform or transfect host cells, and expression of thecDNA of interest can be detected with an antibody to zcytor19polypeptide. Similar techniques can also be applied to the isolation ofgenomic clones.

Cytokine receptor subunits are characterized by a multi-domain structurecomprising an extracellular domain, a transmembrane domain that anchorsthe polypeptide in the cell membrane, and an intracellular domain. Theextracellular domain is typically a ligand-binding domain, and theintracellular domain is typically an effector domain involved in signaltransduction, although ligand-binding and effector functions may resideon separate subunits of a multimeric receptor. The ligand-binding domainmay itself be a multi-domain structure. Multimeric receptors includehomodimers (e.g., PDGF receptor αα and ββ isoforms, erythropoietinreceptor, MPL, and G-CSF receptor), heterodimers whose subunits eachhave ligand-binding and effector domains (e.g., PDGF receptor αβisoform), and multimers having component subunits with disparatefunctions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSFreceptors). Some receptor subunits are common to a plurality ofreceptors. For example, the AIC2B subunit, which cannot bind ligand onits own but includes an intracellular signal transduction domain, is acomponent of IL-3 and GM-CSF receptors. Many cytokine receptors can beplaced into one of four related families on the basis of the structureand function. Hematopoietic receptors, for example, are characterized bythe presence of a domain containing conserved cysteine residues and theWSXWS motif (SEQ ID NO:5). Cytokine receptor structure has been reviewedby Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and Cosman, Cytokine5:95-106, 1993. Under selective pressure for organisms to acquire newbiological functions, new receptor family members likely arise fromduplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members. Thus, thecytokine receptor superfamily is subdivided into several families, forexample, the immunoglobulin family (including CSF-1, MGF, IL-1, and PDGFreceptors); the hematopoietin family (including IL-2 receptor β-subunit,GM-CSF receptor α-subunit, GM-CSF receptor β-subunit; and G-CSF, EPO,IL-3, IL-4, IL-5, IL-6, IL-7, and IL-9 receptors); TNF receptor family(including TNF (p80) TNF (p60) receptors, CD27, CD30, CD40, Fas, and NGFreceptor).

Analysis of the zcytor19 sequence suggests that it is a member of thesame receptor subfamily as the class II cytokine receptors, for example,interferon-gamma, alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No.5,965,704), CRF2-4 (Genbank Accession No. Z17227), DIRSI, zcytor7(commonly owned U.S. Pat. No. 5,945,511) receptors. Receptors in thissubfamily may associate to form homodimers that transduce a signal.Several members of the subfamily (e.g., receptors that bind interferon,IL-10, IL-19, and IL-TIF) combine with a second subunit (termed aβ-subunit) to bind ligand and transduce a signal. Specific β-subunitsassociate with a plurality of specific cytokine receptor subunits. Forexample, with class II cytokine receptors commonly owned zcytor11 (U.S.Pat. No. 5,965,704) and CRF2-4 receptor heterodimerize to bind thecytokine IL-TIF (See, WIPO publication WO 00/24758; Dumontier et al., J.Immunol. 164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med.187:571-578, 1998; Gibbs, V C and Pennica Gene 186:97-101, 1997 (CRF2-4cDNA); Xie, M H et al., J. Biol. Chem. 275: 31335-31339, 2000; andKotenko, S V et al., J. Biol. Chem. manuscript in press M007837200).Moreover, IL-10β receptor may be involved as a receptor for IL-TIF, andit is believed to be synonymous with CRF2-4 (Dumoutier, L. et al., Proc.Nat'l. Acad. Sci. 97:10144-10149, 2000; Liu Y et al, J. Immunol. 152;1821-1829, 1994 (IL-10R cDNA). As such, class II receptor complexes canbe heterodimeric, or multimeric. Thus, monomeric, homodimeric,heterodimeric and multimeric receptors comprising a zcytor19 subunit areencompassed by the present invention.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 represents one allele ofhuman zcytor19 and that allelic variation and alternative splicing areexpected to occur. Allelic variants of this sequence can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the DNA sequence shown inSEQ ID NO:1, SEQ ID NO:18 or SEQ ID NO:20 including those containingsilent mutations and those in which mutations result in amino acidsequence changes, are within the scope of the present invention, as areproteins which are allelic variants of SEQ ID NO:2, SEQ ID NO:19 or SEQID NO:21. cDNAs generated from alternatively spliced mRNAs, which retainthe properties of the zcytor19 polypeptide are included within the scopeof the present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

The present invention also provides isolated zcytor19 polypeptides thatare substantially similar to the polypeptides of SEQ ID NO:2, SEQ IDNO:19 or SEQ ID NO:21 and their orthologs. The term “substantiallysimilar” is used herein to denote polypeptides having at least 70%, morepreferably at least 80%, sequence identity to the sequences shown in SEQID NO:2, SEQ ID NO:19 or SEQ ID NO:21 or their orthologs. Suchpolypeptides will more preferably be at least 90% identical, and mostpreferably 95% or more identical to SEQ ID NO:2, SEQ ID NO:19 or SEQ IDNO:21 or its orthologs.) Percent sequence identity is determined byconventional methods. See, for example, Altschul et al., Bull. Math.Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA 89:10915-10919, 1992. Briefly, two amino acid sequences are alignedto optimize the alignment scores using a gap opening penalty of 10, agap extension penalty of 1, and the “blosum 62” scoring matrix ofHenikoff and Henikoff (ibid.) as shown in Table 3 (amino acids areindicated by the standard one-letter codes). The percent identity isthen calculated as:

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{bmatrix}{{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}} \\{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}} \\{{sequence}\mspace{14mu}{in}\mspace{14mu}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}}\end{bmatrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant zcytor19. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2, SEQ ID NO:19 orSEQ ID NO:21) and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Preferred parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62, with other parameters setas default. These parameters can be introduced into a FASTA program bymodifying the scoring matrix file (“SMATRIX”), as explained in Appendix2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other FASTA programparameters set as default.

The BLOSUM62 table (Table 3) is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed below), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Variant zcytor19 polypeptides or substantially homologous zcytor19polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table4) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides that comprise a sequence that is at least80%, preferably at least 90%, and more preferably 95% or more identicalto the corresponding region of SEQ ID NO:2, SEQ ID NO:19 or SEQ IDNO:21, excluding the tags, extension, linker sequences and the like.Polypeptides comprising affinity tags can further comprise a proteolyticcleavage site between the zcytor19 polypeptide and the affinity tag.Suitable sites include thrombin cleavage sites and factor Xa cleavagesites.

TABLE 4 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a zcytor19 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-zcytor19polypeptide fusions can be expressed in genetically engineered cells toproduce a variety of multimeric zcytor19 analogs. Auxiliary domains canbe fused to zcytor19 polypeptides to target them to specific cells,tissues, or macromolecules (e.g., collagen). A zcytor19 polypeptide canbe fused to two or more moieties, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,Tuan et al., Connective Tissue Research 34:1-9, 1996.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zcytor19 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (e.g.ligand binding and signal transduction) as disclosed below to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites ofligand-receptor, protein-protein or other biological interaction canalso be determined by physical analysis of structure, as determined bysuch techniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904,1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities ofessential amino acids can also be inferred from analysis of homologieswith related receptors.

Determination of amino acid residues that are within regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to, alignment of multiple sequences withhigh amino acid or nucleotide identity and computer analysis usingavailable software (e.g., the Insight II® viewer and homology modelingtools; MSI, San Diego, Calif.), secondary structure propensities, binarypatterns, complementary packing and buried polar interactions (Barton,Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., CurrentOpin. Struct. Biol. 6:3-10, 1996). In general, when designingmodifications to molecules or identifying specific fragmentsdetermination of structure will be accompanied by evaluating activity ofmodified molecules.

Amino acid sequence changes are made in zcytor19 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. For example, when the zcytor19 polypeptide comprises one ormore structural domains, such as Fibronectin Type III domains, changesin amino acid residues will be made so as not to disrupt the domainstructure and geometry and other components of the molecule wherechanges in conformation ablate some critical function, for example,binding of the molecule to its binding partners. The effects of aminoacid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same disulfide bonding patternas the standard molecule folding would be affected. Another well knownand accepted method for measuring folding is circular dichroism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructural similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the zcytor19 protein sequence asshown in SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 can be generated(Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun.Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering 11:153-169,1998). The profile is based on a sliding six-residue window. Buried G,S, and T residues and exposed H, Y, and W residues were ignored. Forexample, in zcytor19, hydrophilic regions include amino acid residues295 through 300 of SEQ ID NO:2; 451 through 456 of SEQ ID NO:2; 301through 306 of SEQ ID NO:2; 244 through 299 of SEQ ID NO:2; and 65through 70 of SEQ ID NO:2. Moreover, one of skill in the art wouldrecognize that zcytor19 hydrophilic regions including antigenicepitope-bearing polypeptides can be predicted by a Jameson-Wolf plot,e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.).

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zcytor19 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include such residues as shown in SEQ ID NO:2.However, Cysteine residues at positions 74, 82, 195, and 217 of SEQ IDNO:2 or SEQ ID NO:19, and corresponding Cys residues in SEQ ID NO:4 arerelatively intolerant of substitution. Moreover, Cysteine residues atpositions 74, 82, of SEQ ID NO:21 are relatively intolerant ofsubstitution.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between class II cytokine receptorfamily members with zcytor19. Using methods such as “FASTA” analysisdescribed previously, regions of high similarity are identified within afamily of proteins and used to analyze amino acid sequence for conservedregions. An alternative approach to identifying a variant zcytor19polynucleotide on the basis of structure is to determine whether anucleic acid molecule encoding a potential variant zcytor19polynucleotide can hybridize to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 asdiscussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule. Such mutagenesis and screeningmethods are routine in the art. See also, Hilton et al., J. Biol. Chem.271:4699 (1996).

The present invention also includes functional fragments of zcytor19polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” zcytor19 or fragment thereof defined herein ischaracterized by its proliferative or differentiating activity, by itsability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-zcytor19 antibody or zcytor19ligand (either soluble or immobilized). Moreover, functional fragmentsalso include the signal peptide, intracellular signaling domain, and thelike. As previously described herein, zcytor19 is characterized by aclass II cytokine receptor structure. Thus, the present inventionfurther provides fusion proteins encompassing: (a) polypeptide moleculescomprising an extracellular domain, cytokine-binding domain, orintracellular domain described herein; and (b) functional fragmentscomprising one or more of these domains. The other polypeptide portionof the fusion protein may be contributed by another class II cytokinereceptor, for example, interferon-gamma, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonlyowned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly ownedU.S. Pat. No. 5,945,511), and the like, or by a non-native and/or anunrelated secretory signal peptide that facilitates secretion of thefusion protein.

Routine deletion analyses of nucleic acid molecules can be performed toobtain functional fragments of a nucleic acid molecule that encodes azcytor19 polypeptide. As an illustration, DNA molecules having thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 orfragments thereof, can be digested with Bal31 nuclease to obtain aseries of nested deletions. These DNA fragments are then inserted intoexpression vectors in proper reading frame, and the expressedpolypeptides are isolated and tested for zcytor19 activity, or for theability to bind anti-zcytor19 antibodies or zcytor19 ligand. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired zcytor19 fragment. Alternatively, particular fragments of azcytor19 polynucleotide can be synthesized using the polymerase chainreaction.

Standard methods for identifying functional domains are well-known tothose of skill in the art. For example, studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1, Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/062045) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zcytor19 DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized zcytor19 receptor polypeptides in host cells.Preferred assays in this regard include cell proliferation assays andbiosensor-based ligand-binding assays, which are described below.Mutagenized DNA molecules that encode active receptors or portionsthereof (e.g., ligand-binding fragments, signaling domains, and thelike) can be recovered from the host cells and rapidly sequenced usingmodern equipment. These methods allow the routine and rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest.

In addition, the proteins of the present invention (or polypeptidefragments thereof) can be joined to other bioactive molecules,particularly cytokine receptors, to provide multi-functional molecules.For example, one or more domains from zcytor19 soluble receptor can bejoined to other cytokine soluble receptors to enhance their biologicalproperties or efficiency of production.

The present invention thus provides a series of novel, hybrid moleculesin which a segment comprising one or more of the domains of zcytor19 isfused to another polypeptide. Fusion is preferably done by splicing atthe DNA level to allow expression of chimeric molecules in recombinantproduction systems. The resultant molecules are then assayed for suchproperties as improved solubility, improved stability, prolongedclearance half-life, improved expression and secretion levels, andpharmacodynamics. Such hybrid molecules may further comprise additionalamino acid residues (e.g. a polypeptide linker) between the componentproteins or polypeptides.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptide fragments or variantsof SEQ ID NO:2 or SEQ ID NO:19 that retain the signal transduction orligand binding activity. For example, one can make a zcytor19 “solublereceptor” by preparing a variety of polypeptides that are substantiallyhomologous to the extracellular cytokine-binding domain (residues 21(Arg) to 226 (Asn) of SEQ ID NO:2 or SEQ ID NO:19), a cytokine-bindingfragment (e.g., residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ IDNO:19; SEQ ID NO:4) or allelic variants or species orthologs thereof)and retain ligand-binding activity of the wild-type zcytor19 protein.Moreover, variant zcytor19 soluble receptors can be isolated. Suchpolypeptides may include additional amino acids from, for example, partor all of the transmembrane and intracellular domains. Such polypeptidesmay also include additional polypeptide segments as generally disclosedherein such as labels, affinity tags, and the like.

For any zcytor19 polypeptide, including variants, soluble receptors, andfusion polypeptides or proteins, one of ordinary skill in the art canreadily generate a fully degenerate polynucleotide sequence encodingthat variant using the information set forth in Tables 1 and 2 above.

The zcytor19 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides, can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zcytor19 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a zcytor19 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zcytor19, or may be derivedfrom another secreted protein (e.g., t-PA) or synthesized de novo. Thesecretory signal sequence is operably linked to the zcytor19 DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid 1 (Met) toamino acid 20 (Gly) of SEQ ID NO:2 or SEQ ID NO:19 is operably linked toanother polypeptide using methods known in the art and disclosed herein.The secretory signal sequence contained in the fusion polypeptides ofthe present invention is preferably fused amino-terminally to anadditional peptide to direct the additional peptide into the secretorypathway. Such constructs have numerous applications known in the art.For example, these novel secretory signal sequence fusion constructs candirect the secretion of a polypeptide fragment or an active component ofa normally non-secreted protein. Such fusions may be used in vivo or invitro to direct peptides through the secretory pathway. Moreover, suchfusion constructs allow for the expression, secretion, and purificationof zcytor19 polypepitde fragments that can be used to inoculate ananimal and generate antibodies, as described herein.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-716, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al, U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection (ATCC), Manassas, Va. Ingeneral, strong transcription promoters are preferred, such as promotersfrom SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. A second method of making recombinant zcytor19 baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system, which utilizes transfervectors, is sold in the Bac-to-Bac™ kit (Life Technologies, Rockville,Md.). This system utilizes a transfer vector, pFastBac1™ (LifeTechnologies) containing a Tn7 transposon to move the DNA encoding thezcytor19 polypeptide into a baculovirus genome maintained in E. coli asa large plasmid called a “bacmid.” See, Hill-Perkins, M. S. and Possee,R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J Gen Virol75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem270:1543-9, 1995. In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C- or N-terminus of theexpressed zcytor19 polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Usinga technique known in the art, a transfer vector containing zcytor19 istransformed into E. coli, and screened for bacmids which contain aninterrupted lacZ gene indicative of recombinant baculovirus. The bacmidDNA containing the recombinant baculovirus genome is isolated, usingcommon techniques, and used to transfect Spodoptera frugiperda cells,e.g. Sf9 cells. Recombinant virus that expresses zcytor19 issubsequently produced. Recombinant viral stocks are made by methodscommonly used in the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf90 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells.Procedures used are generally described in available laboratory manuals(King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;Richardson, C. D., ibid.). Subsequent purification of the zcytor19polypeptide from the supernatant can be achieved using methods describedherein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zcytor19polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

Within one aspect of the present invention, a zcytor19 cytokine receptor(including transmembrane and intracellular domains) is produced by acultured cell, and the cell is used to screen for ligands for thereceptor, including the natural ligand, as well as agonists andantagonists of the natural ligand. To summarize this approach, a cDNA orgene encoding the receptor is combined with other genetic elementsrequired for its expression (e.g., a transcription promoter), and theresulting expression vector is inserted into a host cell. Cells thatexpress the DNA and produce functional receptor are selected and usedwithin a variety of screening systems.

Mammalian cells suitable for use in expressing the novel receptors ofthe present invention and transducing a receptor-mediated signal includecells that express a β-subunit, such as a class II cytokine receptorsubunit, for example, interferon-gamma, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonlyowned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly ownedU.S. Pat. No. 5,945,511) receptors. Such subunits can either naturallybe expressed in the cells, or be co-transfected with zcytor19 receptor.An exemplary cell system for class I cytokine receptors is to use cellsthat express gp130, and cells that co-express gp130 and LIF receptor(Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Pat.No. 5,284,755). In this regard it is generally preferred to employ acell that is responsive to other cytokines that bind to receptors in thesame subfamily, such as IL-6 or LIF, because such cells will contain therequisite signal transduction pathway(s). Preferred cells of this typeinclude BaF3 cells (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), the humanTF-1 cell line (ATCC number CRL-2003) and the DA-1 cell line (Branch etal., Blood 69:1782, 1987; Broudy et al., Blood 75:1622-1626, 1990). Inthe alternative, suitable host cells can be engineered to produce aβ-subunit or other cellular component needed for the desired cellularresponse. For example, the murine cell line BaF3 (Palacios andSteinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol.6: 4133-4135, 1986), a baby hamster kidney (BHK) cell line, or theCTLL-2 cell line (ATCC TIB-214) can be transfected to express individualclass II subunits such as, interferon-gamma, alpha and beta chains andthe interferon-alpha/beta receptor alpha and beta chains, zcytor11(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7(commonly owned U.S. Pat. No. 5,945,511) receptors in addition tozcytor19. It is generally preferred to use a host cell and receptor(s)from the same species, however this approach allows cell lines to beengineered to express multiple receptor subunits from any species,thereby overcoming potential limitations arising from speciesspecificity. In the alternative, species homologs of the human receptorcDNA can be cloned and used within cell lines from the same species,such as a mouse cDNA, in the BaF3 cell line. Cell lines that aredependent upon one hematopoietic growth factor, such as IL-3, can thusbe engineered to become dependent upon a zcytor19 ligand oranti-zcytor19 antibody.

Cells expressing functional zcytor19 are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in the target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycolorimetric assay based on the reduction or metabolic breakdown ofAlymar Blue™ (AccuMed, Chicago, Ill.) or3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, 1983). An alternative assay formatuses cells that are further engineered to express a reporter gene. Thereporter gene is linked to a promoter element that is responsive to thereceptor-linked pathway, e.g, JAK/STAT pathway, and the assay detectsactivation of transcription of the reporter gene. A preferred promoterelement in this regard is a serum response element, SRE (see, forexample, Shaw et al., Cell 56:563-572, 1989). A preferred such reportergene is a luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al., J. Biol. Chem.269:19094-29101, 1994; Schenborn and Goiffin, Promega Notes 41:11,1993). Luciferase assay kits are commercially available from, forexample, Promega Corp., Madison, Wis. Target cell lines of this type canbe used to screen libraries of chemicals, cell-conditioned culturemedia, fungal broths, soil samples, water samples, and the like. Forexample, a bank of cell- or tissue-conditioned media samples can beassayed on a target cell to identify cells that produce ligand. Positivecells are then used to produce a cDNA library in a mammalian cellexpression vector, which is divided into pools, transfected into hostcells, and expressed. Media samples from the transfected cells are thenassayed, with subsequent division of pools, retransfection,subculturing, and re-assay of positive cells to isolate a clonal cellline expressing the ligand. Alternatively, media samples from thetransfected cells can be assayed, with subsequent division of pools,retransfection, and re-assay to isolate a bacterial clone expressing theligand cDNA. Media samples conditioned by kidney, liver, spleen, thymus,other lymphoid tissues, B-cells, T-cells, or leukemia cell lines arepreferred sources of ligand for use in screening procedures.

A natural ligand for zcytor19 can also be identified by mutagenizing acytokine-dependent cell line expressing zcytor19 and culturing it underconditions that select for autocrine growth. See WIPO publication WO95/21930. Within a typical procedure, cells expressing zcytor19 aremutagenized, such as with EMS. The cells are then allowed to recover inthe presence of the required cytokine, then transferred to a culturemedium lacking the cytokine. Surviving cells are screened for theproduction of a ligand for zcytor19, such as by adding soluble receptorpolypeptide comprising the zcytor19 extracellular cytokine-bindingdomain, or cytokine-binding fragment described herein to the culturemedium to compete against the ligand or by assaying conditioned media onwild-type cells compared to transfected cells expressing the zcytor19receptor. Preferred cell lines for use within this method include cellsthat are transfected to express gp130 or gp130 in combination with LIFreceptor. Preferred such host cell lines include transfected CTLL-2cells (Gillis and Smith, Nature 268:154-156, 1977) and transfected BaF3cells.

Moreover, a secretion trap method employing zcytor19 soluble receptorpolypeptide can be used to isolate a zcytor19 ligand (Aldrich, et al,Cell 87: 1161-1169, 1996). A cDNA expression library prepared from aknown or suspected ligand source is transfected into COS-7 cells. ThecDNA library vector generally has an SV40 origin for amplification inCOS-7 cells, and a CMV promoter for high expression. The transfectedCOS-7 cells are grown in a monolayer and then fixed and permeabilized.Tagged or biotin-labeled zcytor19 soluble receptor, described herein, isthen placed in contact with the cell layer and allowed to bind cells inthe monolayer that express an anti-complementary molecule, i.e., azcytor19 ligand. A cell expressing a ligand will thus be bound withreceptor molecules. An anti-tag antibody (anti-Ig for Ig fusions, M2 oranti-FLAG for FLAG-tagged fusions, streptavidin, anti-Glu-Glu tag, andthe like) which is conjugated with horseradish peroxidase (HRP) is usedto visualize these cells to which the tagged or biotin-labeled zcytor19soluble receptor has bound. The HRP catalyzes deposition of a tyramidereagent, for example, tyramide-FITC. A commercially-available kit can beused for this detection (for example, Renaissance TSA-Direct™ Kit; NENLife Science Products, Boston, Mass.). Cells which express zcytor19receptor ligand will be identified under fluorescence microscopy asgreen cells and picked for subsequent cloning of the ligand usingprocedures for plasmid rescue as outlined in Aldrich, et al, supra.,followed by subsequent rounds of secretion trap assay, or conventionalscreening of cDNA library pools, until single clones are identified.

As a receptor, the activity of zcytor19 polypeptide can be measured by asilicon-based biosensor microphysiometer which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. A variety of cellular responses,such as cell proliferation, ion transport, energy production,inflammatory response, regulatory and receptor activation, and the like,can be measured by this method. See, for example, McConnell, H. M. etal., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95, 1998. Themicrophysiometer can be used for assaying eukaryotic, prokaryotic,adherent or non-adherent cells. By measuring extracellular acidificationchanges in cell media over time, the microphysiometer directly measurescellular responses to various stimuli, including agonists, ligands, orantagonists of the zcytor19 polypeptide. Preferably, themicrophysiometer is used to measure responses of a zcytor19-expressingeukaryotic cell, compared to a control eukaryotic cell that does notexpress zcytor19 polypeptide. Zcytor19-expressing eukaryotic cellscomprise cells into which zcytor19 has been transfected or infected viaadenovirus vector, and the like, as described herein, creating a cellthat is responsive to zcytor19-modulating stimuli, or are cellsnaturally expressing zcytor19, such as zcytor19-expressing cells derivedfrom lymphoid, spleen, thymus tissue or PBLs. Differences, measured byan increase or decrease in extracellular acidification, in the responseof cells expressing zcytor19, relative to a control, are a directmeasurement of zcytor19-modulated cellular responses. Moreover, suchzcytor19-modulated responses can be assayed under a variety of stimuli.Also, using the microphysiometer, there is provided a method ofidentifying agonists and antagonists of zcytor19 polypeptide, comprisingproviding cells expressing a zcytor19 polypeptide, culturing a firstportion of the cells in the absence of a test compound, culturing asecond portion of the cells in the presence of a test compound, anddetecting an increase or a decrease in a cellular response of the secondportion of the cells as compared to the first portion of the cells.Antagonists and agonists, including the natural ligand for zcytor19polypeptide, can be rapidly identified using this method.

Additional assays provided by the present invention include the use ofhybrid receptor polypeptides. These hybrid polypeptides fall into twogeneral classes. Within the first class, the intracellular domain ofzcytor19, comprising approximately residues 250 (Lys) to 491 (Arg) ofSEQ ID NO:2 or residues 250 (Lys) to 520 (Arg) of SEQ ID NO:19), isjoined to the ligand-binding domain of a second receptor. It ispreferred that the second receptor be a hematopoietic cytokine receptor,such as mp1 receptor (Souyri et al., Cell 63:1137-1147, 1990). Thehybrid receptor will further comprise a transmembrane domain, which maybe derived from either receptor. A DNA construct encoding the hybridreceptor is then inserted into a host cell. Cells expressing the hybridreceptor are cultured in the presence of a ligand for the binding domainand assayed for a response. This system provides a means for analyzingsignal transduction mediated by zcytor19 while using readily availableligands. This system can also be used to determine if particular celllines are capable of responding to signals transduced by zcytor19. Asecond class of hybrid receptor polypeptides comprise the extracellular(ligand-binding) cytokine-binding domain (residues 21 (Arg) to 226 (Asn)of SEQ ID NO:2 or SEQ ID NO:19), or cytokine-binding fragment (e.g.,residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ ID NO:19; SEQ IDNO:4) with a cytoplasmic domain of a second receptor, preferably acytokine receptor, and a transmembrane domain. The transmembrane domainmay be derived from either receptor. Hybrid receptors of this secondclass are expressed in cells known to be capable of responding tosignals transduced by the second receptor. Together, these two classesof hybrid receptors enable the use of a broad spectrum of cell typeswithin receptor-based assay systems.

Cells found to express a ligand for zcytor19 are then used to prepare acDNA library from which the ligand-encoding cDNA may be isolated asdisclosed above. The present invention thus provides, in addition tonovel receptor polypeptides, methods for cloning polypeptide ligands forthe receptors.

The zcytor19 structure and tissue expression suggests a role in earlyhematopoietic or thymocyte development and immune response regulation.These processes involve stimulation of cell proliferation anddifferentiation in response to the binding of one or more cytokines totheir cognate receptors. In view of the tissue distribution observed forthis receptor, agonists (including the natural ligand) and antagonistshave enormous potential in both in vitro and in vivo applications.Compounds identified as receptor agonists are useful for stimulatingproliferation and development of target cells in vitro and in vivo. Forexample, agonist compounds or antizcytor19 antibodies, are useful ascomponents of defined cell culture media, and may be used alone or incombination with other cytokines and hormones to replace serum that iscommonly used in cell culture. Agonists are thus useful in specificallypromoting the growth and/or development of T-cells, B-cells, and othercells of the lymphoid and myeloid lineages, and hematopoietic cells inculture.

Agonist ligands for zcytor19, or anti-zcytor19 antibodies, may be usefulin stimulating cell-mediated immunity and for stimulating lymphocyteproliferation, such as in the treatment of infections involvingimmunosuppression, including certain viral infections. Additional usesinclude tumor suppression, where malignant transformation results intumor cells that are antigenic. Agonist ligands or anti-zcytor19antibodies could be used to induce cytotoxicity, which may be mediatedthrough activation of effector cells such as T-cells, NK (naturalkiller) cells, or LAK (lymphoid activated killer) cells, or induceddirectly through apoptotic pathways. For example, zcytor19 antibodiescould be used for stimulating cytotoxicity or ADCC on zcytor19-bearingcancer cells. Agonist ligands may also be useful in treating leukopeniasby increasing the levels of the affected cell type, and for enhancingthe regeneration of the T-cell repertoire after bone marrowtransplantation.

Antagonist ligands, compounds, soluble zcytor19 receptors, oranti-zcytor19 antibodies may find utility in the suppression of theimmune system, such as in the treatment of autoimmune diseases,including rheumatoid arthritis, multiple sclerosis, diabetes mellitis,inflammatory bowel disease, Crohn's disease, etc. Immune suppression canalso be used to reduce rejection of tissue or organ transplants andgrafts and to treat T-cell specific leukemias or lymphomas by inhibitingproliferation of the affected cell type.

The present invention contemplates the use of naked anti-zcytor19antibodies (or naked antibody fragments thereof), as well as the use ofimmunoconjugates to effect treatment of various disorders, includingB-cell malignancies and other cancers described herein wherein zcytor19is expressed. Such immunoconjugates as well as anti-zcytor19 antibodiescan be used for stimulating cytotoxicity or ADCC on zcytor19-bearingcancer cells. Immunoconjugates can be prepared using standardtechniques. For example, immunoconjugates can be produced by indirectlyconjugating a therapeutic agent to an antibody component (see, forexample, Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al.,Int. J. Cancer 46:1101-1106 (1990); and Shih et al., U.S. Pat. No.5,057,313). Briefly, one standard approach involves reacting an antibodycomponent having an oxidized carbohydrate portion with a carrier polymerthat has at least one free amine function and that is loaded with aplurality of drug, toxin, chelator, boron addends, or other therapeuticagent. This reaction results in an initial Schiff base (imine) linkage,which can be stabilized by reduction to a secondary amine to form thefinal conjugate.

The carrier polymer can be an aminodextran or polypeptide of at least 50amino acid residues, although other substantially equivalent polymercarriers can also be used. Preferably, the final immunoconjugate issoluble in an aqueous solution, such as mammalian serum, for ease ofadministration and effective targeting for use in therapy. Thus,solubilizing functions on the carrier polymer will enhance the serumsolubility of the final immunoconjugate.

In an alternative approach for producing immunoconjugates comprising apolypeptide therapeutic agent, the therapeutic agent is coupled toaminodextran by glutaraldehyde condensation or by reaction of activatedcarboxyl groups on the polypeptide with amines on the aminodextran.Chelators can be attached to an antibody component to prepareimmunoconjugates comprising radiometals or magnetic resonance enhancers.Illustrative chelators include derivatives of ethylenediaminetetraaceticacid and diethylenetriaminepentaacetic acid. Boron addends, such ascarboranes, can be attached to antibody components by conventionalmethods.

Immunoconjugates can also be prepared by directly conjugating anantibody component with a therapeutic agent. The general procedure isanalogous to the indirect method of conjugation except that atherapeutic agent is directly attached to an oxidized antibodycomponent.

As a further illustration, a therapeutic agent can be attached at thehinge region of a reduced antibody component via disulfide bondformation. For example, the tetanus toxoid peptides can be constructedwith a single cysteine residue that is used to attach the peptide to anantibody component. As an alternative, such peptides can be attached tothe antibody component using a heterobifunctional cross-linker, such asN-succinyl 3-(2-pyridyldithio)proprionate. Yu et al., Int. J. Cancer56:244 (1994). General techniques for such conjugation are well-known inthe art. See, for example, Wong, Chemistry Of Protein Conjugation AndCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: PrinciplesAnd Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering And Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995).

As described above, carbohydrate moieties in the Fc region of anantibody can be used to conjugate a therapeutic agent. However, the Fcregion is absent if an antibody fragment is used as the antibodycomponent of the immunoconjugate. Nevertheless, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofan antibody or antibody fragment. See, for example, Leung et al., J.Immunol. 154:5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995).The engineered carbohydrate moiety is then used to attach a therapeuticagent.

In addition, those of skill in the art will recognize numerous possiblevariations of the conjugation methods. For example, the carbohydratemoiety can be used to attach polyethyleneglycol in order to extend thehalf-life of an intact antibody, or antigen-binding fragment thereof, inblood, lymph, or other extracellular fluids. Moreover, it is possible toconstruct a divalent immunoconjugate by attaching therapeutic agents toa carbohydrate moiety and to a free sulfhydryl group. Such a freesulfhydryl group may be located in the hinge region of the antibodycomponent.

One type of immunoconjugate comprises an antibody component and apolypeptide cytotoxin. An example of a suitable polypeptide cytotoxin isa ribosome-inactivating protein. Type I ribosome-inactivating proteinsare single-chain proteins, while type II ribosome-inactivating proteinsconsist of two nonidentical subunits (A and B chains) joined by adisulfide bond (for a review, see Soria et al., Targeted Diagn. Ther.7:193 (1992)). Useful type I ribosome-inactivating proteins includepolypeptides from Saponaria officinalis (e.g., saporin-1, saporin-2,saporin-3, saporin-6), Momordica charantia (e.g, momordin), Byroniadioica (e.g., bryodin, bryodin-2), Trichosanthes kirilowii (e.g.,trichosanthin, trichokirin), Gelonium multiflorum (e.g., gelonin),Phytolacca americana (e.g., pokeweed antiviral protein, pokeweedantiviral protein-II, pokeweed antiviral protein-S), Phytolaccadodecandra (e.g., dodecandrin, Mirabilis antiviral protein), and thelike. Ribosome-inactivating proteins are described, for example, byWalsh et al., U.S. Pat. No. 5,635,384.

Suitable type II ribosome-inactivating proteins include polypeptidesfrom Ricinus communis (e.g., ricin), Abrus precatorius (e.g., abrin),Adenia digitata (e.g., modeccin), and the like. Since type IIribosome-inactiving proteins include a B chain that binds galactosidesand a toxic A chain that depurinates adensoine, type IIribosome-inactivating protein conjugates should include the A chain.Additional useful ribosome-inactivating proteins include bouganin,clavin, maize ribosome-inactivating proteins, Vaccaria pyramidataribosome-inactivating proteins, nigrine b, basic nigrine 1, ebuline,racemosine b, luffin-a, luffin-b, luffin-S, and otherribosome-inactivating proteins known to those of skill in the art. See,for example, Bolognesi and Stirpe, international publication No.WO98/55623, Colnaghi et al., international publication No. WO97/49726,Hey et al., U.S. Pat. No. 5,635,384, Bolognesi and Stirpe, internationalpublication No. WO95/07297, Arias et al., international publication No.WO94/20540, Watanabe et al., J. Biochem. 106:6 977 (1989); Islam et al.,Agric. Biol. Chem. 55:229 (1991), and Gao et al., FEBS Lett. 347:257(1994).

Analogs and variants of naturally-occurring ribosome-inactivatingproteins are also suitable for the targeting compositions describedherein, and such proteins are known to those of skill in the art.Ribosome-inactivating proteins can be produced using publicly availableamino acid and nucleotide sequences. As an illustration, a nucleotidesequence encoding saporin-6 is disclosed by Lorenzetti et al., U.S. Pat.No. 5,529,932, while Walsh et al., U.S. Pat. No. 5,635,384, describemaize and barley ribosome-inactivating protein nucleotide and amino acidsequences. Moreover, ribosome-inactivating proteins are alsocommercially available.

Additional polypeptide cytotoxins include ribonuclease, DNase I,Staphylococcal enterotoxin-A, diphtheria toxin, Pseudomonas exotoxin,and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641(1986), and Goldenberg, C A -A Cancer Jornal for Clinicians 44:43(1994).

Another general type of useful cytotoxin is a tyrosine kinase inhibitor.Since the activation of proliferation by tyrosine kinases has beensuggested to play a role in the development and progression of tumors,this activation can be inhibited by anti-zcytor19 antibody componentsthat deliver tyrosine kinase inhibitors. Suitable tyrosine kinaseinhibitors include isoflavones, such as genistein(5,7,4′-trihydroxyisoflavone), daidzein (7,4′-dihydroxyisoflavone), andbiochanin A (4-methoxygenistein), and the like. Methods of conjugatingtyrosine inhibitors to a growth factor are described, for example, byUckun, U.S. Pat. No. 5,911,995.

Another group of useful polypeptide cytotoxins includesimmunomodulators. As used herein, the term “immunomodulator” includescytokines, stem cell growth factors, lymphotoxins, co-stimulatorymolecules, hematopoietic factors, and the like, as well as syntheticanalogs of these molecules. Examples of immunomodulators include tumornecrosis factor, interleukins (e.g., interleukin-1(IL-1), IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, and IL-20), colony stimulatingfactors (e.g., granulocyte-colony stimulating factor and granulocytemacrophage-colony stimulating factor), interferons (e.g., interferons-α,-β, -γ, -ω, -ε, and -τ), the stem cell growth factor designated “S1factor,” erythropoietin, and thrombopoietin. Illustrativeimmunomodulator moieties include IL-2, IL-6, IL-10, interferon-□, TNF-□,and the like.

Immunoconjugates that include an immunomodulator provide a means todeliver an immunomodulator to a target cell, and are particularly usefulagainst tumor cells. The cytotoxic effects of immunomodulators are wellknown to those of skill in the art. See, for example, Klegerman et al.,“Lymphokines and Monokines,” in Biotechnology And Pharmacy, Pessuto etal. (eds.), pages 53-70 (Chapman & Hall 1993). As an illustration,interferons can inhibit cell proliferation by inducing increasedexpression of class I histocompatibility antigens on the surface ofvarious cells and thus, enhance the rate of destruction of cells bycytotoxic T lymphocytes. Furthermore, tumor necrosis factors, such astumor necrosis factor-α, are believed to produce cytotoxic effects byinducing DNA fragmentation.

The present invention also includes immunocongugates that comprise anucleic acid molecule encoding a cytotoxin. As an example of thisapproach, Hoganson et al., Human Gene Ther. 9:2565 (1998), describeFGF-2 mediated delivery of a saporin gene by producing anFGF-2-polylysine conjugate which was condensed with an expression vectorcomprising a saporin gene.

Other suitable toxins are known to those of skill in the art.

Conjugates of cytotoxic polypeptides and antibody components can beprepared using standard techniques for conjugating polypeptides. Forexample, Lam and Kelleher, U.S. Pat. No. 5,055,291, describe theproduction of antibodies conjugated with either diphtheria toxinfragment A or ricin toxin. The general approach is also illustrated bymethods of conjugating fibroblast growth factor with saporin, asdescribed by Lappi et al., Biochem. Biophys. Res. Commun. 160:917(1989), Soria et al., Targeted Diagn. Ther. 7:193 (1992), Buechler etal., Eur. J Biochem. 234:706 (1995), Behar-Cohen et al., Invest. Ophthalmol. Vis. Sci. 36:2434 (1995), Lappi and Baird, U.S. Pat. No. 5,191,067,Calabresi et al., U.S. Pat. No. 5,478,804, and Lappi and Baird, U.S.Pat. No. 5,576,288. Also see, Ghetie and Vitteta, “Chemical Constructionof Immunotoxins,” in Drug Targeting: Strategies, Principles, andApplications, Francis and Delgado (Eds.), pages 1-26 (Humana Press, Inc.2000), Hall (Ed.), Immunotoxin Methods and Protocols (Humana Press, Inc.2000), and Newton and Rybak, “Construction of Ribonuclease-AntibodyConjugates for Selective Cytotoxicity,” in Drug Targeting: Strategies,Principles, and Applications, Francis and Delgado (Eds.), pages 27-35(Humana Press, Inc. 2000).

Alternatively, fusion proteins comprising an antibody component and acytotoxic polypeptide can be produced using standard methods. Methods ofpreparing fusion proteins comprising a cytotoxic polypeptide moiety arewell-known in the art of antibody-toxin fusion protein production. Forexample, antibody fusion proteins comprising an interleukin-2 moiety aredescribed by Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al.,Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Natl Acad. Sci. USA93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Hu etal., Cancer Res. 56:4998 (1996). In addition, Yang et al., Hum.Antibodies Hybridomas 6:129 (1995), describe a fusion protein thatincludes an F(ab′)₂ fragment and a tumor necrosis factor alpha moiety.Antibody-Pseudomonas exotoxin A fusion proteins have been described byChaudhary et al., Nature 339:394 (1989), Brinkmann et al., Proc. Nat'lAcad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'l Acad. Sci. USA89:5867 (1992), Friedman et al., J. Immunol. 150:3054 (1993), Wels etal., Int. J. Can. 60:137 (1995), Fominaya et al., J. Biol. Chem.271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996), and Schmidtet al., Int. J. Can. 65:538 (1996). Antibody-toxin fusion proteinscontaining a diphtheria toxin moiety have been described by Kreitman etal., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem. 268:5302(1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), and Vallera etal., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting 1:177(1995), have described an antibody-toxin fusion protein having an RNasemoiety, while Linardou et al., Cell Biophys. 24-25:243 (1994), producedan antibody-toxin fusion protein comprising a DNase I component. Geloninwas used as the toxin moiety in the antibody-toxin fusion protein ofBetter et al., J. Biol. Chem. 270:14951 (1995). As a further example,Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994), reported anantibody-toxin fusion protein comprising Staphylococcal enterotoxin-A.Also see, Newton and Rybak, “Preparation of Recombinant RNaseSingle-Chain Antibody Fusion Proteins,” in Drug Targeting: Strategies,Principles, and Applications, Francis and Delgado (Eds.), pages 77-95(Humana Press, Inc. 2000).

As an alternative to a polypeptide cytotoxin, immunoconjugates cancomprise a radioisotope as the cytotoxic moiety. For example, animmunoconjugate can comprise an anti-zcytor19 antibody component and anα-emitting radioisotope, a β-emitting radioisotope, a γ-emittingradioisotope, an Auger electron emitter, a neutron capturing agent thatemits α-particles or a radioisotope that decays by electron capture.Suitable radioisotopes include ¹⁹⁸Au, ¹⁹⁹Au, ³²P, ³³P, ¹²⁵I, ¹³¹I, ¹²³I,⁹⁰Y, ¹⁸⁶ Re, ¹⁸⁸Re, ⁶⁷Cu, ²¹¹At, ⁴⁷Sc, ¹⁰³Pb, ¹⁰⁹Pd, ²¹²Pb, ⁷¹Ge, ⁷⁷AS,¹⁰⁵Rh, ¹¹³Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os,^(193M)Pt, ¹⁹⁷Hg, and the like.

A radioisotope can be attached to an antibody component directly orindirectly, via a chelating agent. For example, ⁶⁷Cu, which providesβ-particles and γ-rays, can be conjugated to an antibody component usingthe chelating agent, p-bromoacetamido-benzyl-tetraethylaminetetraaceticacid. Chase and Shapiro, “Medical Applications of Radioisotopes,” inGennaro (Ed.), Remington: The Science and Practice of Pharmacy, 19thEdition, pages 843-865 (Mack Publishing Company 1995). As analternative, ⁹⁰Y, which emits an energetic β-particle, can be coupled toan antibody component using diethylenetriaminepentaacetic acid.Moreover, an exemplary suitable method for the direct radiolabeling ofan antibody component with ¹³¹I is described by Stein et al., AntibodyImmunoconj. Radiopharm. 4:703 (1991). Alternatively, boron addends suchas carboranes can be attached to antibody components, using standardtechniques.

Another type of suitable cytotoxin for the preparation ofimmunoconjugates is a chemotherapeutic drug. Illustrativechemotherapeutic drugs include nitrogen mustards, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purineanalogs, antibiotics, epipodophyllotoxins, platinum coordinationcomplexes, and the like. Specific examples of chemotherapeutic drugsinclude methotrexate, doxorubicin, daunorubicin, cytosinarabinoside,cis-platin, vindesine, mitomycin, bleomycin, melphalan, chlorambucil,maytansinoids, calicheamicin, taxol, and the like. Suitablechemotherapeutic agents are described in Remington: The Science andPractice of Pharmacy, 19th Edition (Mack Publishing Co. 1995), and inGoodman And Gilman's The Pharmacological Basis Of Therapeutics, 9th Ed.(MacMillan Publishing Co. 1995). Other suitable chemotherapeutic agentsare known to those of skill in the art.

In another approach, immunoconjugates are prepared by conjugatingphotoactive agents or dyes to an antibody component. Fluorescent andother chromogens, or dyes, such as porphyrins sensitive to visiblelight, have been used to detect and to treat lesions by directing thesuitable light to the lesion. This type of “photoradiation,”“phototherapy,” or “photodynamic” therapy is described, for example, byMew et al., J. Immunol. 130:1473 (1983), Jori et al. (eds.),Photodynamic Therapy Of Tumors And Other Diseases (Libreria Progetto1985), Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986), vanden Bergh, Chem. Britain 22:430 (1986), Hasan et al., Prog. Clin. Biol.Res. 288:471 (1989), Tatsuta et al., Lasers Surg. Med. 9:422 (1989), andPelegrin et al., Cancer 67:2529 (1991).

The approaches described above can also be used to prepare multispecificantibody compositions that comprise an immunoconjugate.

Anti-zcytor19 antibodies and multispecific antibody compositions can beused to modulate the immune system by preventing the binding of zcytor19ligands with endogenous zcytor19 receptors. Such antibodies can beadministered to any subject in need of treatment, and the presentinvention contemplates both veterinary and human therapeutic uses.Illustrative subjects include mammalian subjects, such as farm animals,domestic animals, and human patients.

Multispecific antibody compositions and dual reactive antibodies thatbind zcytor19 can be used for the treatment of autoimmune diseases, Bcell cancers, immunomodulation, and other pathologies (e.g., ITCP, Tcell-mediated diseases, cattleman's disease, autoimmune disease,myelodysplastic syndrome, and the like), renal diseases, graftrejection, and graft versus host disease. The antibodies of the presentinvention can be targeted to specifically regulate B cell responsesduring the immune response. Additionally, the antibodies of the presentinvention can be used to modulate B cell development, antigenpresentation by B cells, antibody production, and cytokine production.

Antagonistic anti-zcytor19 antibodies can be useful to neutralize theeffects of zcytor19 ligands for treating B cell lymphomas and leukemias,chronic or acute lymphocytic leukemia, myelomas such as multiplemyeloma, plasma cytomas, and lymphomas such as non-Hodgkins lymphoma,for which an increase in zcytor19 ligand polypeptides is associated, orwhere zcytor19 ligand is a survival factor or growth factor.Anti-zcytor19 antibodies can also be used to treat Epstein Barrvirus-associated lymphomas arising in immunocompromised patients (e.g.,AIDS or organ transplant).

Anti-zcytor19 antibodies that induce a signal by binding with zcytor19may inhibit the growth of lymphoma and leukemia cells directly viainduction of signals that lead to growth inhibition, cell cycle arrest,apoptosis, or tumor cell death. Zcytor19 antibodies that initiate asignal are preferred antibodies to directly inhibit or kill cancercells. In addition, agonistic anti-zcytor19 monoclonal antibodies mayactivate normal B cells and promote an anticancer immune response.Anti-zcytor19 antibodies may directly inhibit the growth of leukemias,lymphomas, and multiple myelomas, and the antibodies may engage immuneeffector functions. Anti-zcytor19 monoclonal antibodies may enableantibody-dependent cellular cytotoxicity, complement dependentcytotoxicity, and phagocytosis.

zcytor19 ligand may be expressed in neutrophils, monocytes, dendriticcells, and activated monocytes. In certain autoimmune disorders (e.g.,myasthenia gravis, and rheumatoid arthritis), B cells might exacerbateautoimmunity after activation by zcytor19 ligand. Immunosuppressantproteins that selectively block the action of B-lymphocytes would be ofuse in treating disease. Autoantibody production is common to severalautoimmune diseases and contributes to tissue destruction andexacerbation of disease. Autoantibodies can also lead to the occurrenceof immune complex deposition complications and lead to many symptoms ofsystemic lupus erythematosus, including kidney failure, neuralgicsymptoms and death. Modulating antibody production independent ofcellular response would also be beneficial in many disease states. Bcells have also been shown to play a role in the secretion ofarthritogenic immunoglobulins in rheumatoid arthritis. As such,inhibition of zcytor19 ligand antibody production would be beneficial intreatment of autoimmune diseases such as myasthenia gravis andrheumatoid arthritis. Immunosuppressant therapeutics such asanti-zcytor19 antibodies that selectively block or neutralize the actionof B-lymphocytes would be useful for such purposes.

The invention provides methods employing anti-zcytor19 antibodies, ormultispecific antibody compositions, for selectively blocking orneutralizing the actions of B-cells in association with end stage renaldiseases, which may or may not be associated with autoimmune diseases.Such methods would also be useful for treating immunologic renaldiseases. Such methods would be would be useful for treatingglomerulonephritis associated with diseases such as membranousnephropathy, IgA nephropathy or Berger's Disease, IgM nephropathy,Goodpasture's Disease, post-infectious glomerulonephritis,mesangioproliferative disease, chronic lymphocytic leukemia,minimal-change nephrotic syndrome. Such methods would also serve astherapeutic applications for treating secondary glomerulonephritis orvasculitis associated with such diseases as lupus, polyarteritis,Henoch-Schonlein, Scleroderma, HIV-related diseases, amyloidosis orhemolytic uremic syndrome. The methods of the present invention wouldalso be useful as part of a therapeutic application for treatinginterstitial nephritis or pyelonephritis associated with chronicpyelonephritis, analgesic abuse, nephrocalcinosis, nephropathy caused byother agents, nephrolithiasis, or chronic or acute interstitialnephritis.

The present invention also provides methods for treatment of renal orurological neoplasms, multiple myelomas, lymphomas, leukemias, lightchain neuropathy, or amyloidosis.

The invention also provides methods for blocking or inhibiting activatedB cells using anti-zcytor19 antibodies, or multispecific antibodycompositions, for the treatment of asthma and other chronic airwaydiseases such as bronchitis and emphysema.

Also provided are methods for inhibiting or neutralizing a T cellresponse using anti-zcytor19 antibodies, or multispecific antibodycompositions, for immunosuppression, in particular for such therapeuticuse as for graft-versus-host disease and graft rejection. Moreover,anti-zcytor19 antibodies, or multispecific antibody compositions, wouldbe useful in therapeutic protocols for treatment of such autoimmunediseases as insulin dependent diabetes mellitus (IDDM), multiplesclerosis, rheumatoid arthritis, systemic lupus erythematosus,inflammatory bowel disease (IBD), and Crohn's Disease. Methods of thepresent invention would have additional therapeutic value for treatingchronic inflammatory diseases, in particular to lessen joint pain,swelling, anemia and other associated symptoms as well as treatingseptic shock.

B cell responses are important in fighting infectious diseases includingbacterial, viral, protozoan and parasitic infections. Antibodies againstinfectious microorganisms can immobilize the pathogen by binding toantigen followed by complement mediated lysis or cell mediated attack.Agonistic, or signaling, anti-zcytor19 antibodies may serve to boost thehumoral response and would be a useful therapeutic for individuals atrisk for an infectious disease or as a supplement to vaccination.

Well established animal models are available to test in vivo efficacy ofanti-zcytor19 antibodies, or multispecific antibody compositions, of thepresent invention in certain disease states. As an illustration,anti-zcytor19 antibodies can be tested in vivo in a number of animalmodels of autoimmune disease, such as MRL-lpr/lpr or NZB×NZW F1 congenicmouse strains which serve as a model of systemic lupus erythematosus.Such animal models are known in the art.

Offspring of a cross between New Zealand Black (NZB) and New ZealandWhite (NZW) mice develop a spontaneous form of systemic lupuserythematosus that closely resembles systemic lupus erythematosus inhumans. The offspring mice, known as NZBW begin to develop IgMautoantibodies against T-cells at one month of age, and by 5-7 months ofage, Ig anti-DNA autoantibodies are the dominant immunoglobulin.Polyclonal B-cell hyperactivity leads to overproduction ofautoantibodies. The deposition of these autoantibodies, particularlyones directed against single stranded DNA is associated with thedevelopment of glomerulonephritis, which manifests clinically asproteinuria, azotemia, and death from renal failure. Kidney failure isthe leading cause of death in mice affected with spontaneous systemiclupus erythematosus, and in the NZBW strain, this process is chronic andobliterative. The disease is more rapid and severe in females thanmales, with mean survival of only 245 days as compared to 406 days forthe males. While many of the female mice will be symptomatic(proteinuria) by 7-9 months of age, some can be much younger or olderwhen they develop symptoms. The fatal immune nephritis seen in the NZBWmice is very similar to the glomerulonephritis seen in human systemiclupus erythematosus, making this spontaneous murine model useful fortesting of potential systemic lupus erythematosus therapeutics.

Murine models of experimental allergic encephalomyelitis have been usedas tools to investigate both the mechanisms of immune-mediated disease,and methods of potential therapeutic intervention. The model resembleshuman multiple sclerosis, and produces demyelination as a result ofT-cell activation to neural proteins such as myelin basic protein, orproteolipid protein. Inoculation with antigen leads to induction ofCD4+, class II MHC-restricted T-cells. Changes in the protocol forexperimental allergic encephalomyelitis can produce acute,chronic-relapsing, or passive-transfer variants of the model.

In the collagen-induced arthritis model, mice develop chronicinflammatory arthritis, which closely resembles human rheumatoidarthritis. Since collagen-induced arthritis shares similar immunologicaland pathological features with rheumatoid arthritis, this makes it anideal model for screening potential human anti-inflammatory compounds.Another advantage in using the collagen-induced arthritis model is thatthe mechanisms of pathogenesis are known. The T and B cell epitopes ontype II collagen have been identified, and various immunological(delayed-type hypersensitivity and anti-collagen antibody) andinflammatory (cytokines, chemokines, and matrix-degrading enzymes)parameters relating to immune-mediating arthritis have been determined,and can be used to assess test compound efficacy in the models.

Myasthenia gravis is another autoimmune disease for which murine modelsare available. Myasthenia gravis is a disorder of neuromusculartransmission involving the production of autoantibodies directed againstthe nicotinic acetylcholine receptor. Myasthenia gravis is acquired orinherited with clinical features including abnormal weakness and fatigueon exertion. A mouse model of myasthenia gravis have been established.Experimental autoimmune myasthenia gravis is an antibody mediateddisease characterized by the presence of antibodies to acetylcholinereceptor. These antibodies destroy the receptor leading to defectiveneuromuscular electrical impulses, resulting in muscle weakness. In theexperimental autoimmune myasthenia gravis model, mice are immunized withthe nicotinic acetylcholine receptor. Clinical signs of myastheniagravis become evident weeks after the second immunization. Experimentalautoimmune myasthenia gravis is evaluated by several methods includingmeasuring serum levels of acetylcholine receptor antibodies byradioimmunoassay, measuring muscle acetylcholine receptor, orelectromyography.

Generally, the dosage of administered anti-zcytor19 antibodies, ormultispecific antibody compositions, will vary depending upon suchfactors as the subject's age, weight, height, sex, general medicalcondition and previous medical history. As an illustration,anti-zcytor19 antibodies, or multispecific antibody compositions, can beadministered at low protein doses, such as 20 to 100 milligrams proteinper dose, given once, or repeatedly. Alternatively, anti-zcytor19antibodies, or multispecific antibody compositions, can be administeredin doses of 30 to 90 milligrams protein per dose, or 40 to 80 milligramsprotein per dose, or 50 to 70 milligrams protein per dose, although alower or higher dosage also may be administered as circumstancesdictate.

Administration of antibody components to a subject can be intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous,intrapleural, intrathecal, by perfusion through a regional catheter, orby direct intralesional injection. When administering therapeuticproteins by injection, the administration may be by continuous infusionor by single or multiple boluses. Additional routes of administrationinclude oral, mucosal-membrane, pulmonary, and transcutaneous.

A pharmaceutical composition comprising an anti-zcytor19 antibody, orbispecific antibody components, can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thetherapeutic proteins are combined in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

For purposes of therapy, anti-zcytor19 antibodies, or bispecificantibody components, and a pharmaceutically acceptable carrier areadministered to a patient in a therapeutically effective amount. Acombination of anti-zcytor19 antibodies, or bispecific antibodycomponents, and a pharmaceutically acceptable carrier is said to beadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient. For example, an agent used to treatinflammation is physiologically significant if its presence alleviatesthe inflammatory response. As another example, an agent used to inhibitthe growth of tumor cells is physiologically significant if theadministration of the agent results in a decrease in the number of tumorcells, decreased metastasis, a decrease in the size of a solid tumor, orincreased necrosis of a tumor.

A pharmaceutical composition comprising anti-zcytor19 antibodies, orbispecific antibody components, can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239(1997); Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems,Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (PlenumPress 1997); Yewey et al., “Delivery of Proteins from a ControlledRelease Injectable Implant,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).

Those of skill in the art can devise various pharmaceutical compositionsusing standard techniques. See, for example, Lieberman et al., (Eds.),Pharmaceutical Dosage Forms: Tablets, Vol. 1, 2nd Edition (MarcelDekker, Inc. 1989), Lieberman et al., (Eds.), Pharmaceutical DosageForms: Tablets, Vol. 2, 2nd Edition (Marcel Dekker, Inc. 1990),Lieberman et al., (Eds.), Pharmaceutical Dosage Forms: Tablets, Vol. 3,2nd Edition (Marcel Dekker, Inc. 1990), Lieberman et al., (Eds.),Pharmaceutical Dosage Forms: Disperse Systems, Vol. 1, 2nd Edition(Marcel Dekker, Inc. 1996), Lieberman et al., (Eds.), PharmaceuticalDosage Forms: Disperse Systems, Vol. 2, 2nd Edition (Marcel Dekker, Inc.1996), Lieberman et al., (Eds.), Pharmaceutical Dosage Forms: DisperseSystems, Vol. 3, 2nd Edition (Marcel Dekker, Inc. 1998), Avis et al.,(Eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, 2ndEdition (Marcel Dekker, Inc. 1991), Lieberman et al., (Eds.),Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 2, 2nd Edition(Marcel Dekker, Inc. 1992), and Avis et al., (Eds.), PharmaceuticalDosage Forms: Parenteral Medications, Vol. 3, 2nd Edition (MarcelDekker, Inc. 1993).

As another example, liposomes provide a means to deliver anti-zcytor19antibodies, or bispecific antibody components, to a subjectintravenously, intraperitoneally, intrathecally, intramuscularly,subcutaneously, or via oral administration, inhalation, or intranasaladministration. Liposomes are microscopic vesicles that consist of oneor more lipid bilayers surrounding aqueous compartments (see, generally,Bakker-Woudenberg et al., Eur. J Clin. Microbiol. Infect. Dis. 12(Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), and Ranade,“Site-Specific Drug Delivery Using Liposomes as Carriers,” in DrugDelivery Systems, Ranade and Hollinger (Eds.), pages 3-24 (CRC Press1995)). Liposomes are similar in composition to cellular membranes andas a result, liposomes can be administered safely and are biodegradable.Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and liposomes can vary in size with diameters rangingfrom 0.02 μm to greater than 10 μm. A variety of agents can beencapsulated in liposomes: hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s) (see,for example, Machy et al., Liposomes In Cell Biology And Pharmacology(John Libbey 1987), and Ostro et al., American J. Hosp. Pharm. 46:1576(1989)). Moreover, it is possible to control the therapeuticavailability of the encapsulated agent by varying liposome size, thenumber of bilayers, lipid composition, as well as the charge and surfacecharacteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

As an alternative to administering liposomes that comprise ananti-zcytor19 antibody component, target cells can be prelabeled withbiotinylated anti-zcytor19 antibodies. After plasma elimination of freeantibody, streptavidin-conjugated liposomes are administered. Thisgeneral approach is described, for example, by Harasym et al., Adv. DrugDeliv. Rev. 32:99 (1998). Such an approach can also be used to preparemultispecific antibody compositions.

Polypeptides comprising an anti-zcytor19 antibody component, orbispecific antibody components, can be encapsulated within liposomes, orattached to the exterior of liposomes, using standard techniques (see,for example, Anderson et al., Infect. Immun. 31:1099 (1981), Wassef etal., Meth. Enzymol. 149:124 (1987), Anderson et al., Cancer Res. 50:1853(1990), Cohen et al., Biochim. Biophys. Acta 1063:95 (1991), Alving etal. “Preparation and Use of Liposomes in Immunological Studies,” inLiposome Technology, 2nd Edition, Vol. III, Gregoriadis (Ed.), page 317(CRC Press 1993), and Ansell et al., “Antibody Conjugation Methods forActive Targeting of Liposomes,” in Drug Targeting: Strategies,Principles, and Applications, Francis and Delgado (Eds.), pages 51-68(Humana Press, Inc. 2000)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim. Biophys. Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (Eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modified antibodycomponents, in which an antibody component is linked with a polymer.Typically, the polymer is water soluble so that an antibody componentdoes not precipitate in an aqueous environment, such as a physiologicalenvironment. An example of a suitable polymer is one that has beenmodified to have a single reactive group, such as an active ester foracylation, or an aldehyde for alkylation. In this way, the degree ofpolymerization can be controlled. An example of a reactive aldehyde ispolyethylene glycol propionaldehyde, or mono-(C₁-C₁₀)alkoxy, or aryloxyderivatives thereof (see, for example, Harris, et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Moreover, amixture of polymers can be used to produce conjugates with antibodycomponents.

Suitable water-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C₁-C₁₀)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. A conjugate can also comprise a mixture of suchwater-soluble polymers.

As an illustration, a polyalkyl oxide moiety can be attached to theN-terminus of antibody component. PEG is one suitable polyalkyl oxide.As an illustration, an antibody component can be modified with PEG, aprocess known as “PEGylation.” PEGylation of an antibody component canbe carried out by any of the PEGylation reactions known in the art (see,for example, EP 0 154 316, Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico,Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J. Hematol68:1 (1998)). For example, PEGylation can be performed by an acylationreaction or by an alkylation reaction with a reactive polyethyleneglycol molecule. In an alternative approach, antibody componentconjugates are formed by condensing activated PEG, in which a terminalhydroxy or amino group of PEG has been replaced by an activated linker(see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with an antibody component. An example of an activatedPEG ester is PEG esterified to N-hydroxysuccinimide. As used herein, theterm “acylation” includes the following types of linkages between anantibody component and a water soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated anti-zcytor19antibody components by acylation will typically comprise the steps of(a) reacting an antibody component with PEG (such as a reactive ester ofan aldehyde derivative of PEG) under conditions whereby one or more PEGgroups attach to the antibody component, and (b) obtaining the reactionproduct(s). Generally, the optimal reaction conditions for acylationreactions will be determined based upon known parameters and desiredresults. For example, the larger the ratio of PEG:antibody component,the greater the percentage of polyPEGylated antibody component product.

The product of PEGylation by acylation is typically a polyPEGylatedantibody component product, wherein the lysine ε-amino groups arePEGylated via an acyl linking group. An example of a connecting linkageis an amide. Typically, the resulting antibody component will be atleast 95% mono-, di-, or tri-pegylated, although some species withhigher degrees of PEGylation may be formed depending upon the reactionconditions. PEGylated species can be separated from unconjugatedantibody component using standard purification methods, such asdialysis, ultrafiltration, ion exchange chromatography, affinitychromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with antibody component in the presence of a reducingagent. PEG groups can be attached to the polypeptide via a —CH₂—NHgroup.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer antibody component conjugate molecule can comprise the stepsof: (a) reacting an antibody component with a reactive PEG underreductive alkylation conditions at a pH suitable to permit selectivemodification of the α-amino group at the amino terminus of the antibodycomponent, and (b) obtaining the reaction product(s). The reducing agentused for reductive alkylation should be stable in aqueous solution andpreferably be able to reduce only the Schiff base formed in the initialprocess of reductive alkylation. Preferred reducing agents includesodium borohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer antibodycomponent conjugates, the reductive alkylation reaction conditions arethose which permit the selective attachment of the water soluble polymermoiety to the N-terminus of the antibody component. Such reactionconditions generally provide for pKa differences between the lysineamino groups and the α-amino group at the N-terminus. The pH alsoaffects the ratio of polymer to protein to be used. In general, if thepH is lower, a larger excess of polymer to protein will be desiredbecause the less reactive the N-terminal α-group, the more polymer isneeded to achieve optimal conditions. If the pH is higher, thepolymer:antibody component need not be as large because more reactivegroups are available. Typically, the pH will fall within the range of 3to 9, or 3 to 6.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997)).

Polypeptide cytotoxins can also be conjugated with a soluble polymerusing the above methods either before or after conjugation to anantibody component. Soluble polymers can also be conjugated withantibody fusion proteins.

Naked anti-zcytor19 antibodies, or antibody fragments, can besupplemented with immunoconjugate or antibody fusion proteinadministration. In one variation, naked anti-zcytor19 antibodies (ornaked antibody fragments) are administered with low-dose radiolabeledanti-zcytor19 antibodies or antibody fragments. As a second alternative,naked anti-zcytor19 antibodies (or antibody fragments) are administeredwith low-dose radiolabeled anti-zcytor19 antibodies-cytokineimmunoconjugates. As a third alternative, naked anti-zcytor19 antibodies(or antibody fragments) are administered with anti-zcytor19-cytokineimmunoconjugates that are not radiolabeled. With regard to “low doses”of ¹³¹I-labeled immunoconjugates, a preferable dosage is in the range of15 to 40 mCi, while the most preferable range is 20 to 30 mCi. Incontrast, a preferred dosage of ⁹⁰Y-labeled immunoconjugates is in therange from 10 to 30 mCi, while the most preferable range is 10 to 20mCi. Similarly, bispecific antibody components can be supplemented withimmunoconjugate or antibody fusion protein administration.

Immunoconjugates having a boron addend-loaded carrier for thermalneutron activation therapy will normally be effected in similar ways.However, it will be advantageous to wait until non-targetedimmunoconjugate clears before neutron irradiation is performed.Clearance can be accelerated using an antibody that binds to theimmunoconjugate. See U.S. Pat. No. 4,624,846 for a description of thisgeneral principle.

The present invention also contemplates a method of treatment in whichimmunomodulators are administered to prevent, mitigate or reverseradiation-induced or drug-induced toxicity of normal cells, andespecially hematopoietic cells. Adjunct immunomodulator therapy allowsthe administration of higher doses of cytotoxic agents due to increasedtolerance of the recipient mammal. Moreover, adjunct immunomodulatortherapy can prevent, palliate, or reverse dose-limiting marrow toxicity.Examples of suitable immunomodulators for adjunct therapy includegranulocyte-colony stimulating factor, granulocyte macrophage-colonystimulating factor, thrombopoietin, IL-1, IL-3, IL-12, and the like. Themethod of adjunct immunomodulator therapy is disclosed by Goldenberg,U.S. Pat. No. 5,120,525.

The efficacy of anti-zcytor19 antibody therapy can be enhanced bysupplementing naked antibody components with immunoconjugates and otherforms of supplemental therapy described herein. In such multimodalregimens, the supplemental therapeutic compositions can be administeredbefore, concurrently or after administration of naked anti-zcytor19antibodies. Multimodal therapies of the present invention furtherinclude immunotherapy with naked anti-zcytor19 antibody componentssupplemented with administration of anti-zcytor19 immunoconjugates. Inanother form of multimodal therapy, subjects receive naked anti-zcytor19antibodies and standard cancer chemotherapy.

The efficacy of an antibody component as a vaccine can be enhanced byconjugating the antibody component to a soluble immunogenic carrierprotein. Suitable carrier proteins include tetanus toxin/toxoid, NTHihigh molecular weight protein, diphtheria toxin/toxoid, detoxified P.aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid,Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen,hepatitis B core antigen, rotavirus VP 7 protein, respiratory syncytialvirus F and G protein, and the like. Methods of preparing conjugatedvaccines are known to those of skill in the art. See, for example, Cruseand Lewis (Eds.), Conjugate Vaccines (S. Karger Publishing 1989), andO'Hagan (Ed.), Vaccine Adjuvants (Humana Press, Inc. 2000). Avaccination composition can also include an adjuvant. Examples ofsuitable adjuvants include aluminum hydroxide and lipid. Methods offormulating vaccine compositions are well-known to those of ordinaryskill in the art. See, for example, Rola, “Immunizing Agents andDiagnostic Skin Antigens,” in Remington: The Science and Practice ofPharmacy, 19th Edition, Gennaro (Ed.), pages 1417-1433 (Mack PublishingCompany 1995).

Pharmaceutical compositions may be supplied as a kit comprising acontainer that comprises anti-zcytor19 antibody components, orbispecific antibody components. Therapeutic molecules can be provided inthe form of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of an anti-zcytor19antibody component. Such a kit may further comprise written informationon indications and usage of the pharmaceutical composition. Moreover,such information may include a statement that the composition iscontraindicated in patients with known hypersensitivity to exogenousantibodies.

Zcytor19 polypeptides, such as soluble zcytor19 receptors, may also beused within diagnostic systems for the detection of circulating levelsof ligand. Within a related embodiment, antibodies or other agents thatspecifically bind to zcytor19 receptor polypeptides can be used todetect circulating receptor polypeptides. Elevated or depressed levelsof ligand or receptor polypeptides may be indicative of pathologicalconditions, including cancer. Soluble receptor polypeptides maycontribute to pathologic processes and can be an indirect marker of anunderlying disease. For example, elevated levels of soluble IL-2receptor in human serum have been associated with a wide variety ofinflammatory and neoplastic conditions, such as myocardial infarction,asthma, myasthenia gravis, rheumatoid arthritis, acute T-cell leukemia,B-cell lymphomas, chronic lymphocytic leukemia, colon cancer, breastcancer, and ovarian cancer (Heaney et al., Blood 87:847-857, 1996).Similarly, as zcytor19 is expressed in B-cell leukemia cells, anincrease of zcytor19 expression can even serve as a marker of anunderlying disease, such as leukemia.

A ligand-binding polypeptide of a zcytor19 receptor, or “solublereceptor,” can be prepared by expressing a truncated DNA encoding thezcytor19 extracellular cytokine-binding domain (residues 21 (Arg) to 226(Asn) of SEQ ID NO:2 or SEQ ID NO:19), cytokine-binding fragment (e.g.,residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ ID NO:19; SEQ IDNO:4), the soluble version of zcytor19 variant, or the correspondingregion of a non-human receptor. It is preferred that the extracellulardomain be prepared in a form substantially free of transmembrane andintracellular polypeptide segments. Moreover, ligand-binding polypeptidefragments within the zcytor19 cytokine-binding domain, described above,can also serve as zcytor19 soluble receptors for uses described herein.To direct the export of a receptor polypeptide from the host cell, thereceptor DNA is linked to a second DNA segment encoding a secretorypeptide, such as a t-PA secretory peptide or a zcytor19 secretorypeptide. To facilitate purification of the secreted receptorpolypeptide, a C-terminal extension, such as a poly-histidine tag,Glu-Glu tag peptide, substance P, Flag™ peptide (Hopp et al.,Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., NewHaven, Conn.) or another polypeptide or protein for which an antibody orother specific binding agent is available, can be fused to the receptorpolypeptide.

In an alternative approach, a receptor extracellular domain can beexpressed as a fusion with immunoglobulin heavy chain constant regions,typically an F_(c) fragment, which contains two constant region domainsand lacks the variable region. Such fusions are typically secreted asmultimeric molecules wherein the F_(c) portions are disulfide bonded toeach other and two receptor polypeptides are arrayed in close proximityto each other. Fusions of this type can be used to affinity purify thecognate ligand from solution, as an in vitro assay tool, to blocksignals in vitro by specifically titrating out ligand, and asantagonists in vivo by administering them parenterally to bindcirculating ligand and clear it from the circulation. To purify ligand,a zcytor19-Ig chimera is added to a sample containing the ligand (e.g.,cell-conditioned culture media or tissue extracts) under conditions thatfacilitate receptor-ligand binding (typically near-physiologicaltemperature, pH, and ionic strength). The chimera-ligand complex is thenseparated by the mixture using protein A, which is immobilized on asolid support (e.g., insoluble resin beads). The ligand is then elutedusing conventional chemical techniques, such as with a salt or pHgradient. In the alternative, the chimera itself can be bound to a solidsupport, with binding and elution carried out as above. Collectedfractions can be re-fractionated until the desired level of purity isreached.

Moreover, zcytor19 soluble receptors can be used as a “ligand sink,”i.e., antagonist, to bind ligand in vivo or in vitro in therapeutic orother applications where the presence of the ligand is not desired. Forexample, in cancers that are expressing large amount of bioactivezcytor19 ligand, zcytor19 soluble receptors can be used as a directantagonist of the ligand in vivo, and may aid in reducing progressionand symptoms associated with the disease. Moreover, zcytor19 solublereceptor can be used to slow the progression of cancers thatover-express zcytor19 receptors, by binding ligand in vivo that wouldotherwise enhance proliferation of those cancers. Similar in vitroapplications for a zcytor19 soluble receptor can be used, for instance,as a negative selection to select cell lines that grow in the absence ofzcytor19 ligand.

Moreover, zcytor19 soluble receptor can be used in vivo or in diagnosticapplications to detect zcytor19 ligand-expressing cancers in vivo or intissue samples. For example, the zcytor19 soluble receptor can beconjugated to a radio-label or fluorescent label as described herein,and used to detect the presence of the ligand in a tissue sample usingan in vitro ligand-receptor type binding assay, or fluorescent imagingassay. Moreover, a radiolabeled zcytor19 soluble receptor could beadministered in vivo to detect ligand-expressing solid tumors through aradio-imaging method known in the art. Similarly, zcytor19polynucleotides, polypeptides, anti-zcytor19 andibodies, or peptidebinding fragments can be used to detect zcytor19 receptor expressingcancers. In a preferred embodiment zcytor19 polynucleotides,polypeptides, anti-zcytor19 andibodies, or peptide binding fragments canbe used to detect leukemias, more preferably B-cell leukemias, and mostpreferably pre-B-cell acute lymphoblastic leukemia.

Differentiation is a progressive and dynamic process, beginning withpluripotent stem cells and ending with terminally differentiated cells.Pluripotent stem cells that can regenerate without commitment to alineage express a set of differentiation markers that are lost whencommitment to a cell lineage is made. Progenitor cells express a set ofdifferentiation markers that may or may not continue to be expressed asthe cells progress down the cell lineage pathway toward maturation.Differentiation markers that are expressed exclusively by mature cellsare usually functional properties such as cell products, enzymes toproduce cell products, and receptors. The stage of a cell population'sdifferentiation is monitored by identification of markers present in thecell population. Myocytes, osteoblasts, adipocytes, chrondrocytes,fibroblasts and reticular cells are believed to originate from a commonmesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988).Markers for mesenchymal stem cells have not been well identified (Owenet al., J. of Cell Sci. 87:731-738, 1987), so identification is usuallymade at the progenitor and mature cell stages. The novel polypeptides ofthe present invention may be useful for studies to isolate mesenchymalstem cells and myocyte or other progenitor cells, both in vivo and exvivo.

There is evidence to suggest that factors that stimulate specific celltypes down a pathway towards terminal differentiation ordedifferentiation affect the entire cell population originating from acommon precursor or stem cell. Thus, the present invention includesstimulating or inhibiting the proliferation of lymphoid cells,hematopoietic cells and endothelial cells. Thus molecules of the presentinvention, such as soluble zcytor19 receptors, cytokine-bindingfragments, anti-zcytor19 antibodies, sense and antisense polynucleotidesmay have use in inhibiting tumor cells, and particularly lymphoid,hematopoietic, prostate, endothelial, and thyroid tumor cells.

Assays measuring differentiation include, for example, measuring cellmarkers associated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Watt, FASEB,5:281-284, 1991; Francis, Differentiation 57:63-75, 1994; Raes, Adv.Anim. Cell Biol. Technol. Bioprocesses, 161-171, 1989; all incorporatedherein by reference). Alternatively, zcytor19 polypeptide itself canserve as an additional cell-surface or secreted marker associated withstage-specific expression of a tissue. As such, direct measurement ofzcytor19 polypeptide, or its loss of expression in a tissue as itdifferentiates, can serve as a marker for differentiation of tissues.Moreover, since zcytor19 is specifically-expressed in pre-B cell acutelymphoblastic leukemia cells, as well as several other cancers asdescribed herein. As such, one of skill in the art would recognize thatthe polynucleotides, polypeptides and antibodies of the presentinvention can be used as a marker for these cancers.

Similarly, direct measurement of zcytor19 polypeptide, or its loss ofexpression in a tissue can be determined in a tissue or cells as theyundergo tumor progression. Increases in invasiveness and motility ofcells, or the gain or loss of expression of zcytor19 in a pre-cancerousor cancerous condition, in comparison to normal tissue, can serve as adiagnostic for transformation, invasion and metastasis in tumorprogression. As such, knowledge of a tumor's stage of progression ormetastasis will aid the physician in choosing the most proper therapy,or aggressiveness of treatment, for a given individual cancer patient.Methods of measuring gain and loss of expression (of either mRNA orprotein) are well known in the art and described herein and can beapplied to zcytor19 expression. For example, appearance or disappearanceof polypeptides that regulate cell motility can be used to aid diagnosisand prognosis of prostate cancer (Banyard, J. and Zetter, B. R., Cancerand Metast. Rev. 17:449-458, 1999). As an effector of cell motility, oras a B-cell tumor-specific marker, zcytor19 gain or loss of expressionmay serve as a diagnostic for lymphoid, B-cell, endothelial,hematopoietic and other cancers. Moreover, analogous to the prostatespecific antigen (PSA), as a naturally-expressed tissue-specific marker,increased levels of zcytor19 polypeptides, or anti-zcytor19 antibodiesin a patient, relative to a normal control can be indicative of diseasein normal tissues where zcytor19 is expressed (See, e.g., Mulders, T MT, et al., Eur. J. Surgical Oncol. 16:37-41, 1990). Moreover, wherezcytor19 expression appears to be restricted to specific normal humantissues, lack of zcytor19 expression in those tissues or strong zcytor19expression in non-specific tissues would serve as a diagnostic of anabnormality in the cell or tissue type, of invasion or metastasis ofcancerous tissues into non-cancerous tissue, and could aid a physicianin directing further testing or investigation, or aid in directingtherapy. As zcytor19 is expressed in esophagus, liver, ovary, rectum,stomach, and uterus tumors, and melanoma, disgnostic probes haveparticular use in diagnosing and identifying tissues from these cancers.

In addition, as zcytor19 is tissue-specific, polynucleotide probes,anti-zcytor19 antibodies, and detection the presence of zcytor19polypeptides in tissue can be used to assess whether a specific tissueis present, for example, after surgery involving the excision of adiseased or cancerous tissues in which zcytor19 is expressed. As such,the polynucleotides, polypeptides, and antibodies of the presentinvention can be used as an aid to determine whether all tissue isexcised after surgery, for example, after surgery for cancer. In suchinstances, it is especially important to remove all potentially diseasedtissue to maximize recovery from the cancer, and to minimize recurrence.Preferred embodiments include fluorescent, radiolabeled, orcalorimetrically labeled anti-zcytor19 antibodies and zcytor19polypeptide binding partners, that can be used histologically or insitu. Specific tissues in which zcytor19 is exporessed are disclosedherein.

Moreover, the activity and effect of zcytor19 on tumor progression andmetastasis can be measured in vivo. Several syngeneic mouse models havebeen developed to study the influence of polypeptides, compounds orother treatments on tumor progression. In these models, tumor cellspassaged in culture are implanted into mice of the same strain as thetumor donor. The cells will develop into tumors having similarcharacteristics in the recipient mice, and metastasis will also occur insome of the models. Appropriate tumor models for our studies include theLewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.CRL-6323), amongst others. These are both commonly used tumor lines,syngeneic to the C57BL6 mouse, that are readily cultured and manipulatedin vitro. Tumors resulting from implantation of either of these celllines are capable of metastasis to the lung in C57BL6 mice. The Lewislung carcinoma model has recently been used in mice to identify aninhibitor of angiogenesis (O'Reilly M S, et al. Cell 79: 315-328,1994).C57BL6/J mice are treated with an experimental agent either throughdaily injection of recombinant protein, agonist or antagonist or a onetime injection of recombinant adenovirus. Three days following thistreatment, 10⁵ to 10⁶ cells are implanted under the dorsal skin.Alternatively, the cells themselves may be infected with recombinantadenovirus, such as one expressing zcytor19, before implantation so thatthe protein is synthesized at the tumor site or intracellularly, ratherthan systemically. The mice normally develop visible tumors within 5days. The tumors are allowed to grow for a period of up to 3 weeks,during which time they may reach a size of 1500-1800 mm³ in the controltreated group. Tumor size and body weight are carefully monitoredthroughout the experiment. At the time of sacrifice, the tumor isremoved and weighed along with the lungs and the liver. The lung weighthas been shown to correlate well with metastatic tumor burden. As anadditional measure, lung surface metastases are counted. The resectedtumor, lungs and liver are prepared for histopathological examination,immunohistochemistry, and in situ hybridization, using methods known inthe art and described herein. The influence of the expressed polypeptidein question, e.g., zcytor19, on the ability of the tumor to recruitvasculature and undergo metastasis can thus be assessed. In addition,aside from using adenovirus, the implanted cells can be transientlytransfected with zcytor19. Use of stable zcytor19 transfectants as wellas use of induceable promoters to activate zcytor19 expression in vivoare known in the art and can be used in this system to assess zcytor19induction of metastasis. Moreover, purified zcytor19 or zcytor19conditioned media can be directly injected in to this mouse model, andhence be used in this system. For general reference see, O'Reilly M S,et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models ofLiver Metastasis. Invasion Metastasis 14:349-361, 1995.

The activity of zcytor19 and its derivatives (conjugates) on growth anddissemination of tumor cells derived from human hematologic malignanciescan also be measured in vivo in a mouse Xenograft model. Several mousemodels have been developed in which human tumor cells are implanted intoimmunodeficient mice, collectively referred to as xenograft models. SeeCattan, A R and Douglas, E Leuk. Res. 18:513-22, 1994; and Flavell, D J,Hematological Oncology 14:67-82, 1996. The characteristics of thedisease model vary with the type and quantity of cells delivered to themouse. Typically, the tumor cells will proliferate rapidly and can befound circulating in the blood and populating numerous organ systems.Therapeutic strategies appropriate for testing in such a model includeantibody induced toxicity, ligand-toxin conjugates or cell-basedtherapies. The latter method, commonly referred to adoptiveimmunotherapy, involves treatment of the animal with components of thehuman immune system (i.e. lymphocytes, NK cells) and may include ex vivoincubation of cells with zcytor19 or other immunomodulatory agents.

The mRNA corresponding to this novel DNA shows expression in lymphoidtissues, including pre-B cell acute lymphoblastic leukemia, bone marrow,and may be expressed in spleen, lymph nodes, and peripheral bloodleukocytes. These data indicate a role for the zcytor19 receptor inleukemia, including B-cell leukemia, proliferation, differentiation,and/or activation of immune cells, and suggest a role in development andregulation of immune responses. The data also suggest that theinteraction of zcytor19 with its ligand may stimulate proliferation anddevelopment of myeloid cells and may, like cytoikine receptors IL-2,IL-6, LIF, IL-11 and OSM (Baumann et al., J. Biol. Chem. 268:8414-8417,1993), induce acute-phase protein synthesis in hepatocytes.

It is preferred to purify the polypeptides of the present invention to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified polypeptideis substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Expressed recombinant zcytor19 polypeptides (or zcytor19 chimeric orfusion polypeptides) can be purified using fractionation and/orconventional purification methods and media. Ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their biochemical, structural, and biologicalproperties. For example, immobilized metal ion adsorption (IMAC)chromatography can be used to purify histidine-rich proteins, includingthose comprising polyhistidine tags. Briefly, a gel is first chargedwith divalent metal ions to form a chelate (Sulkowski, Trends inBiochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to thismatrix with differing affinities, depending upon the metal ion used, andwill be eluted by competitive elution, lowering the pH, or use of strongchelating agents. Other methods of purification include purification ofglycosylated proteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), Acad. Press, San Diego, 1990,pp.529-39). Within additional embodiments of the invention, a fusion ofthe polypeptide of interest and an affinity tag (e.g., maltose-bindingprotein, an immunoglobulin domain) may be constructed to facilitatepurification.

Moreover, using methods described in the art, polypeptide fusions, orhybrid zcytor19 proteins, are constructed using regions or domains ofthe inventive zcytor19 in combination with those of other human cytokinereceptor family proteins, or heterologous proteins (Sambrook et al.,ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5,1994, and references therein). These methods allow the determination ofthe biological importance of larger domains or regions in a polypeptideof interest. Such hybrids may alter reaction kinetics, binding,constrict or expand the substrate specificity, or alter tissue andcellular localization of a polypeptide, and can be applied topolypeptides of unknown structure.

Fusion polypeptides or proteins can be prepared by methods known tothose skilled in the art by preparing each component of the fusionprotein and chemically conjugating them. Alternatively, a polynucleotideencoding one or more components of the fusion protein in the properreading frame can be generated using known techniques and expressed bythe methods described herein. For example, part or all of a domain(s)conferring a biological function may be swapped between zcytor19 of thepresent invention with the functionally equivalent domain(s) fromanother cytokine family member. Such domains include, but are notlimited to, the secretory signal sequence, extracellular cytokinebinding domain, cytokine binding fragment, fibronectin type III domains,transmembrane domain, and intracellular signaling domain, as disclosedherein. Such fusion proteins would be expected to have a biologicalfunctional profile that is the same or similar to polypeptides of thepresent invention or other known family proteins, depending on thefusion constructed. Moreover, such fusion proteins may exhibit otherproperties as disclosed herein.

Standard molecular biological and cloning techniques can be used to swapthe equivalent domains between the zcytor19 polypeptide and thosepolypeptides to which they are fused. Generally, a DNA segment thatencodes a domain of interest, e.g., a zcytor19 domain described herein,is operably linked in frame to at least one other DNA segment encodingan additional polypeptide (for instance a domain or region from anothercytokine receptor, such as, interferon-gamma, alpha and beta chains andthe interferon-alpha/beta receptor alpha and beta chains, zcytor11(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7(commonly owned U.S. Pat. No. 5,945,511), or other class II cytokinereceptor), and inserted into an appropriate expression vector, asdescribed herein. Generally DNA constructs are made such that theseveral DNA segments that encode the corresponding regions of apolypeptide are operably linked in frame to make a single construct thatencodes the entire fusion protein, or a functional portion thereof. Forexample, a DNA construct would encode from N-terminus to C-terminus afusion protein comprising a signal polypeptide followed by a cytokinebinding domain, followed by a transmembrane domain, followed by anintracellular signaling domain. Such fusion proteins can be expressed,isolated, and assayed for activity as described herein. Moreover, suchfusion proteins can be used to express and secrete fragments of thezcytor19 polypeptide, to be used, for example to inoculate an animal togenerate anti-zcytor19 antibodies as described herein. For example asecretory signal sequence can be operably linked to extracellularcytokine binding domain, cytokine binding fragment, individualfibronectin type III domains, transmembrane domain, and intracellularsignaling domain, as disclosed herein, or a combination thereof (e.g.,operably linked polypeptides comprising a fibronectin III domainattached to a linker, or zcytor19 polypeptide fragments describedherein), to secrete a fragment of zcytor19 polypeptide that can bepurified as described herein and serve as an antigen to be inoculatedinto an animal to produce anti-zcytor19 antibodies, as described herein.

Zcytor19 polypeptides or fragments thereof may also be prepared throughchemical synthesis. Zcytor19 polypeptides may be monomers or multimers;glycosylated or non-glycosylated; pegylated or non-pegylated; and may ormay not include an initial methionine amino acid residue.

Polypeptides of the present invention can also be synthesized byexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. Methods for synthesizingpolypeptides are well known in the art. See, for example, Merrifield, J.Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595,1970. After the entire synthesis of the desired peptide on a solidsupport, the peptide-resin is with a reagent which cleaves thepolypeptide from the resin and removes most of the side-chain protectinggroups. Such methods are well established in the art.

The activity of molecules of the present invention can be measured usinga variety of assays that measure cell differentiation and proliferation.Such assays are well known in the art and described herein.

Proteins of the present invention are useful for example, in treatinglymphoid, immune, inflammatory, spleenic, blood or bone disorders, andcan be measured in vitro using cultured cells or in vivo byadministering molecules of the present invention to the appropriateanimal model. For instance, host cells expressing a zcytor19 solublereceptor polypeptide can be embedded in an alginate environment andinjected (implanted) into recipient animals. Alginate-poly-L-lysinemicroencapsulation, permselective membrane encapsulation and diffusionchambers are a means to entrap transfected mammalian cells or primarymammalian cells. These types of non-immunogenic “encapsulations” permitthe diffusion of proteins and other macromolecules secreted or releasedby the captured cells to the recipient animal. Most importantly, thecapsules mask and shield the foreign, embedded cells from the recipientanimal's immune response. Such encapsulations can extend the life of theinjected cells from a few hours or days (naked cells) to several weeks(embedded cells). Alginate threads provide a simple and quick means forgenerating embedded cells.

The materials needed to generate the alginate threads are known in theart. In an exemplary procedure, 3% alginate is prepared in sterile H₂O,and sterile filtered. Just prior to preparation of alginate threads, thealginate solution is again filtered. An approximately 50% cellsuspension (containing about 5×10⁵ to about 5×10⁷ cells/ml) is mixedwith the 3% alginate solution. One ml of the alginate/cell suspension isextruded into a 100 mM sterile filtered CaCl₂ solution over a timeperiod of ˜15 min, forming a “thread”. The extruded thread is thentransferred into a solution of 50 mM CaCl₂, and then into a solution of25 mM CaCl₂. The thread is then rinsed with deionized water beforecoating the thread by incubating in a 0.01% solution of poly-L-lysine.Finally, the thread is rinsed with Lactated Ringer's Solution and drawnfrom solution into a syringe barrel (without needle). A large boreneedle is then attached to the syringe, and the thread isintraperitoneally injected into a recipient in a minimal volume of theLactated Ringer's Solution.

An in vivo approach for assaying proteins of the present inventioninvolves viral delivery systems. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: (i) adenovirus can accommodate relatively large DNA inserts;(ii) can be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) can be used with a large number of differentpromoters including ubiquitous, tissue specific, and regulatablepromoters. Also, because adenoviruses are stable in the bloodstream,they can be administered by intravenous injection.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene has been deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell (the human 293 cell line is exemplary). Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an E1 genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined.

Moreover, adenoviral vectors containing various deletions of viral genescan be used in an attempt to reduce or eliminate immune responses to thevector. Such adenoviruses are E1 deleted, and in addition containdeletions of E2A or E4 (Lusky, M. et al., J. Virol. 72:2022-2032, 1998;Raper, S. E. et al., Human Gene Therapy 9:671-679, 1998). In addition,deletion of E2b is reported to reduce immune responses (Amalfitano, A.et al., J. Virol. 72:926-933, 1998). Moreover, by deleting the entireadenovirus genome, very large inserts of heterologous DNA can beaccommodated. Generation of so called “gutless” adenoviruses where allviral genes are deleted are particularly advantageous for insertion oflarge inserts of heterologous DNA. For review, see Yeh, P. andPerricaudet, M., FASEB J. 11:615-623, 1997.

The adenovirus system can also be used for protein production in vitro.By culturing adenovirus-infected non-293 cells under conditions wherethe cells are not rapidly dividing, the cells can produce proteins forextended periods of time. For instance, BHK cells are grown toconfluence in cell factories, then exposed to the adenoviral vectorencoding the secreted protein of interest. The cells are then grownunder serum-free conditions, which allows infected cells to survive forseveral weeks without significant cell division. Alternatively,adenovirus vector infected 293 cells can be grown as adherent cells orin suspension culture at relatively high cell density to producesignificant amounts of protein (See Gamier et al., Cytotechnol.15:145-55, 1994). With either protocol, an expressed, secretedheterologous protein can be repeatedly isolated from the cell culturesupernatant, lysate, or membrane fractions depending on the dispositionof the expressed protein in the cell. Within the infected 293 cellproduction protocol, non-secreted proteins may also be effectivelyobtained.

In view of the tissue distribution observed for zcytor19, agonists(including the natural ligand/substrate/cofactor/etc.) and antagonistshave enormous potential in both in vitro and in vivo applications.Compounds identified as zcytor19 agonists are useful for stimulatinggrowth of immune and hematopoietic cells in vitro and in vivo. Forexample, zcytor19 soluble receptors, and agonist compounds are useful ascomponents of defined cell culture media, and may be used alone or incombination with other cytokines and hormones to replace serum that iscommonly used in cell culture. Agonists are thus useful in specificallypromoting the growth and/or development of T-cells, B-cells, and othercells of the lymphoid and myeloid lineages in culture. Moreover,zcytor19 soluble receptor, agonist, or antagonist may be used in vitroin an assay to measure stimulation of colony formation from isolatedprimary bone marrow cultures. Such assays are well known in the art.

Antagonists are also useful as research reagents for characterizingsites of ligand-receptor interaction. Inhibitors of zcytor19 activity(zcytor19 antagonists) include anti-zcytor19 antibodies and solublezcytor19 receptors, as well as other peptidic and non-peptidic agents(including ribozymes).

Zcytor19 can also be used to identify modulators (e.g, antagonists) ofits activity. Test compounds are added to the assays disclosed herein toidentify compounds that inhibit the activity of zcytor19. In addition tothose assays disclosed herein, samples can be tested for inhibition ofzcytor19 activity within a variety of assays designed to measurezcytor19 binding, oligomerization, or the stimulation/inhibition ofzcytor19-dependent cellular responses. For example, zcytor19-expressingcell lines can be transfected with a reporter gene construct that isresponsive to a zcytor19-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a zcytor19-DNA response element operably linked to a geneencoding an assay detectable protein, such as luciferase. DNA responseelements can include, but are not limited to, cyclic AMP responseelements (CRE), hormone response elements (HRE) insulin response element(IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) andserum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989).Cyclic AMP response elements are reviewed in Roestler et al., J. Biol.Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4(8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell56:335-44; 1989. Candidate compounds, solutions, mixtures or extracts orconditioned media from various cell types are tested for the ability toenhance the activity of zcytor19 receptor as evidenced by a increase inzcytor19 stimulation of reporter gene expression. Assays of this typewill detect compounds that directly stimulate zcytor19 signaltransduction activity through binding the receptor or by otherwisestimulating part of the signal cascade. As such, there is provided amethod of identifying agonists of zcytor19 polypeptide, comprisingproviding cells responsive to a zcytor19 polypeptide, culturing a firstportion of the cells in the absence of a test compound, culturing asecond portion of the cells in the presence of a test compound, anddetecting a increase in a cellular response of the second portion of thecells as compared to the first portion of the cells. Moreover a thirdcell, containing the reporter gene construct described above, but notexpressing zcytor19 receptor, can be used as a control cell to assessnon-specific, or non-zcytor19-mediated, stimulation of the reporter.Agonists, including the natural ligand, are therefore useful tostimulate or increase zcytor19 polypeptide function.

A zcytor19 ligand-binding polypeptide, such as the extracellular domainor cytokine binding domain disclosed herein, can also be used forpurification of ligand. The polypeptide is immobilized on a solidsupport, such as beads of agarose, cross-linked agarose, glass,cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingligand are passed through the column one or more times to allow ligandto bind to the receptor polypeptide. The ligand is then eluted usingchanges in salt concentration, chaotropic agents (guanidine HCl), or pHto disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument may beadvantageously employed (e.g., BIAcore™, Pharmacia Biosensor,Piscataway, N.J.; or SELDI™ technology, Ciphergen, Inc., Palo Alto,Calif.). Such receptor, antibody, member of a complement/anti-complementpair or fragment is immobilized onto the surface of a receptor chip. Useof this instrument is disclosed by Karlsson, J. Immunol. Methods145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63,1993. A receptor, antibody, member or fragment is covalently attached,using amine or sulfhydryl chemistry, to dextran fibers that are attachedto gold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-672, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

Zcytor19 polypeptides can also be used to prepare antibodies that bindto zcytor19 epitopes, peptides or polypeptides. The zcytor19 polypeptideor a fragment thereof serves as an antigen (immunogen) to inoculate ananimal and elicit an immune response. One of skill in the art wouldrecognize that antigenic, epitope-bearing polypeptides contain asequence of at least 6, preferably at least 9, and more preferably atleast 15 to about 30 contiguous amino acid residues of a zcytor19polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21).Polypeptides comprising a larger portion of a zcytor19 polypeptide,i.e., from 30 to 100 residues up to the entire length of the amino acidsequence are included. Antigens or immunogenic epitopes can also includeattached tags, adjuvants and carriers, as described herein. Suitableantigens include the zcytor19 polypeptide encoded by SEQ ID NO:2 fromamino acid number 21 (Arg) to amino acid number 491 (Arg), or acontiguous 9 to 471 amino acid fragment thereof. Suitable antigens alsoinclude the zcytor19 polypeptide encoded by SEQ ID NO:19 from amino acidnumber 21 (Arg) to amino acid number 520 (Arg), or a contiguous 9 to 500amino acid fragment thereof; and the truncated soluble zcytor19polypeptide encoded by SEQ ID NO:21 from amino acid number 21 (Arg) toamino acid number 211 (Ser), or a contiguous 9 to 191 amino acidfragment thereof. Preferred peptides to use as antigens are theextracellular cytokine binding domain, cytokine binding fragment,fibronectin type III domains, intracellular signaling domain, or otherdomains and motifs disclosed herein, or a combination thereof; andzcytor19 hydrophilic peptides such as those predicted by one of skill inthe art from a hydrophobicity plot, determined for example, from aHopp/Woods hydrophilicity profile based on a sliding six-residue window,with buried G, S, and T residues and exposed H, Y, and W residuesignored. Zcytor19 hydrophilic peptides include peptides comprising aminoacid sequences selected from the group consisting of: (1) residues 295through 300 of SEQ ID NO:2; (2) residues 451 through 456 of SEQ ID NO:2;(3) residues 301 through 306 of SEQ ID NO:2; (4) residues 294 through299 of SEQ ID NO:2; and (5) residues 65 through 70 of SEQ ID NO:2. Inaddition, zcytor19 antigenic epitopes as predicted by a Jameson-Wolfplot, e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.)are suitable antigens. In addition, conserved motifs, and variableregions between conserved motifs of zcytor19 are suitable antigens.Antibodies generated from this immune response can be isolated andpurified as described herein. Methods for preparing and isolatingpolyclonal and monoclonal antibodies are well known in the art. See, forexample, Current Protocols in Immunology, Cooligan, et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats with a zcytor19 polypeptide or a fragment thereof. Theimmunogenicity of a zcytor19 polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of zcytor19 or aportion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication WO98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to zcytor19 protein orpeptide, and selection of antibody display libraries in phage or similarvectors (for instance, through use of immobilized or labeled zcytor19protein or peptide). Genes encoding polypeptides having potentialzcytor19 polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using thezcytor19 sequences disclosed herein to identify proteins which bind tozcytor19. These “binding peptides” which interact with zcytor19polypeptides can be used for tagging cells, e.g., such as those in whichzcytor19 is specifically expressed; for isolating homolog polypeptidesby affinity purification; they can be directly or indirectly conjugatedto drugs, toxins, radionuclides and the like. These binding peptides canalso be used in analytical methods such as for screening expressionlibraries and neutralizing activity. The binding peptides can also beused for diagnostic assays for determining circulating levels ofzcytor19 polypeptides; for detecting or quantitating soluble zcytor19polypeptides as marker of underlying pathology or disease. These bindingpeptides can also act as zcytor19 “antagonists” to block zcytor19binding and signal transduction in vitro and in vivo. Theseanti-zcytor19 binding peptides would be useful for inhibiting the actionof a ligand that binds with zcytor19.

Antibodies are considered to be specifically binding if: 1) they exhibita threshold level of binding activity, and 2) they do not significantlycross-react with related polypeptide molecules. A threshold level ofbinding is determined if anti-zcytor19 antibodies herein bind to azcytor19 polypeptide, peptide or epitope with an affinity at least10-fold greater than the binding affinity to control (non-zcytor19)polypeptide. It is preferred that the antibodies exhibit a bindingaffinity (Ka) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, morepreferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ or greater.The binding affinity of an antibody can be readily determined by one ofordinary skill in the art, for example, by Scatchard analysis(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).

Whether anti-zcytor19 antibodies do not significantly cross-react withrelated polypeptide molecules is shown, for example, by the antibodydetecting zcytor19 polypeptide but not known related polypeptides usinga standard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are those disclosed in the prior art, such asknown orthologs, and paralogs, and similar known members of a proteinfamily (e.g., class II cytokine receptors, for example,interferon-gamma, alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No.5,965,704), CRF2-4, DIRSI, zcytor7 (commonly owned U.S. Pat. No.5,945,511) receptors). Screening can also be done using non-humanzcytor19, and zcytor19 mutant polypeptides. Moreover, using routinemethods, antibodies can be “screened against” known relatedpolypeptides, to isolate a population that specifically binds to thezcytor19 polypeptides. For example, antibodies raised to zcytor19 areadsorbed to related polypeptides adhered to insoluble matrix; antibodiesspecific to zcytor19 will flow through the matrix under the properbuffer conditions. Screening allows isolation of polyclonal andmonoclonal antibodies non-crossreactive to known closely relatedpolypeptides (Antibodies: A Laboratorv Manual, Harlow and Lane (eds.),Cold Spring Harbor Laboratory Press, 1988; Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995). Screening and isolation of specificantibodies is well known in the art. See, Fundamental Immunology, Paul(eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98,1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W.(eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol.2: 67-101, 1984. Specifically binding anti-zcytor19 antibodies can bedetected by a number of methods in the art, and disclosed below.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to zcytor19 proteins orpeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutantzcytor19 protein or polypeptide.

Antibodies to zcytor19 may be used for tagging cells that expresszcytor19; for isolating zcytor19 by affinity purification; fordiagnostic assays for determining circulating levels of zcytor19polypeptides; for detecting or quantitating soluble zcytor19 as markerof underlying pathology or disease; for detecting or quantitating in ahistologic, biopsy, or tissue sample zcytor19 receptor as marker ofunderlying pathology or disease; for stimulating cytotoxicity or ADCC onzcytor19-bearing cancer cells; in analytical methods employing FACS; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzcytor19 activity in vitro and in vivo. Suitable direct tags or labelsinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like; indirect tags or labels may feature use of biotin-avidin orother complement/anti-complement pairs as intermediates. Antibodiesherein may also be directly or indirectly conjugated to drugs, toxins,radionuclides and the like, and these conjugates used for in vivodiagnostic or therapeutic applications. Moreover, antibodies to zcytor19or fragments thereof may be used in vitro to detect denatured zcytor19or fragments thereof in assays, for example, Western Blots or otherassays known in the art.

Antibodies to zcytor19 are useful for tagging cells that express thereceptor and assaying Zcytor19 expression levels, for affinitypurification, within diagnostic assays for determining circulatinglevels of soluble receptor polypeptides, analytical methods employingfluorescence-activated cell sorting. Divalent antibodies may be used asagonists to mimic the effect of a zcytor19 ligand.

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications. For instance, antibodiesor binding polypeptides which recognize zcytor19 of the presentinvention can be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (i.e., a zcytor19receptor). More specifically, anti-zcytor19 antibodies, or bioactivefragments or portions thereof, can be coupled to detectable or cytotoxicmolecules and delivered to a mammal having cells, tissues or organs thatexpress the zcytor19 molecule. A preferred use of such conjugatedantibodies is to target the drug to cancers that express the zcytor19receptor. For example, such antibodies can be used to target lymphoid,B-cell, and pre-B-cell acute lymphoblastic leukemia cancers, andesophagus, liver, ovary, rectum, stomach, and uterus tumors, andmelanoma.

Suitable detectable molecules may be directly or indirectly attached topolypeptides that bind zcytor19 (“binding polypeptides,” includingbinding peptides disclosed above), antibodies, or bioactive fragments orportions thereof. Suitable detectable molecules include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Binding polypeptides or antibodies may also be conjugatedto cytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the binding polypeptide orantibody portion. For these purposes, biotin/streptavidin is anexemplary complementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues,e.g., such as those specific tissues and tumors wherein zcytor19 isexpressed). Alternatively, if the binding polypeptide has multiplefunctional domains (i.e., an activation domain or a ligand bindingdomain, plus a targeting domain), a fusion protein including only thetargeting domain may be suitable for directing a detectable molecule, acytotoxic molecule or a complementary molecule to a cell or tissue typeof interest. In instances where the fusion protein including only asingle domain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates.

Similarly, in another embodiment, zcytor19 binding polypeptide-cytokineor antibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues (for example, blood, lymphoid, colon, and bonemarrow cancers, or other cancers described herein wherin zcytor19 isexpressed), if the binding polypeptide-cytokine or anti-zcytor19antibody targets the hyperproliferative cell (See, generally, Hornick etal., Blood 89:4437-47, 1997). They described fusion proteins enabletargeting of a cytokine to a desired site of action, thereby providingan elevated local concentration of cytokine. Suitable anti-zcytor19antibodies target an undesirable cell or tissue (i.e., a tumor or aleukemia), and the fused cytokine mediates improved target cell lysis byeffector cells. Suitable cytokines for this purpose include interleukin2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), forinstance.

Alternatively, zcytor19 binding polypeptide or antibody fusion proteinsdescribed herein can be used for enhancing in vivo killing of targettissues by directly stimulating a zcytor19-modulated apoptotic pathway,resulting in cell death of hyperproliferative cells expressing zcytor19.

The bioactive binding polypeptide or antibody conjugates describedherein can be delivered orally, intravenously, intraarterially orintraductally, or may be introduced locally at the intended site ofaction.

Moreover, anti-zcytor19 antibodies and binding frangments can be usedfor tagging and sorting cells that specifically-express Zcytor19, suchas bone marrow and thyroid cells, and other cells, described herein.Such methods of cell tagging and sorting are well known in the art (see,e.g., “Molecular Biology of the Cell”, 3^(rd) Ed., Albert, B. et al.(Garland Publishing, London & New York, 1994). One of skill in the artwould recognize the importance of separating cell tissue types to studycells, and the use of antibodies to separate specific cell tissue types.Basically, antibodies that bind to the surface of a cell type arecoupled to various matrices such as collagen, polysaccharide beads, orplastic to form an affinity surface to which only cells recognized bythe antibodies will adhere. The bound cells are then recovered byconventional techniques. Other methods involve separating cells by flowcytometry, or using a fluorescence-activated cell sorter (FACS). In thistechnique one labels cells with antibodies that are coupled to afluorescent dye. The labeled cells are then separated from unlabeledcells in a FACS machine. In FACS sorting individual cells traveling insingle file pass through a laser beam and the fluorescence of each cellis measured. Slightly further down-stream, tiny droplets, mostcontaining either one or no cells, are formed by a vibrating nozzle. Thedroplets containing a single cell are automatically give a positive ornegative charge at the moment of formation, depending on whether thecell they contain is fluorescent, and then deflected by a strongelectric field into an appropriate container. Such machines can select 1cell in 1000 and sort about 5000 cells each second. This produces auniform population of cells for cell culture.

One of skill in the art would recognize that the antibodies to theZcytor19 polypeptides of the present invention are useful, because notall tissue types express the Zcytor19 receptor and because it isimportant that biologists be able to separate specific cell types forfurther study and/or therapeutic re-implantation into the body. This isparticularly relevant in cells such as immune cells, wherein zcytor19 isexpressed.

Four-helix bundle cytokines that bind to cytokine receptors as well asother proteins produced by activated lymphocytes play an importantbiological role in cell differentiation, activation, recruitment andhomeostasis of cells throughout the body. Therapeutic utility includestreatment of diseases which require immune regulation includingautoimmune diseases, such as, rheumatoid arthritis, multiple sclerosis,myasthenia gravis, systemic lupus erythomatosis and diabetes. Zcytor19receptor antagonists or agonists, including zcytor19 soluble receptors,anti-receptor antibodies, and the natural ligand, may be important inthe regulation of inflammation, and therefore would be useful intreating rheumatoid arthritis, asthma, ulcerative colitis, inflammatorybowel disease, Crohn's disease, and sepsis. There may be a role ofzcytor19 antagonists or agonists, including soluble receptors,anti-receptor antibodies and the natural ligand, in mediatingtumorgenesis, and therefore would be useful in the treatment of cancer.Zcytor19 antagonists or agonists, including soluble receptorsanti-receptor antibodies and the natural ligand, may be a potentialtherapeutic in suppressing the immune system which would be importantfor reducing graft rejection or in prevention of graft vs. host disease.

Alternatively, zcytor19 antagonists or agonists, including solublereceptors, anti-zcytor19 receptor antibodies and the natural ligand mayactivate the immune system which would be important in boosting immunityto infectious diseases, treating immunocompromised patients, such asHIV+ patient, or in improving vaccines. In particular, zcytor19antagonists or agonists, including soluble receptors, anti-receptorantibodies, and the natural ligand can modulate, stimulate or expand NKcells, or their progenitors, and would provide therapeutic value intreatment of viral infection, and as an anti-neoplastic factor. NK cellsare thought to play a major role in elimination of metastatic tumorcells and patients with both metastases and solid tumors have decreasedlevels of NK cell activity (Whiteside et. al., Curr. Top. Microbiol.Immunol. 230:221-244, 1998).

Polynucleotides encoding zcytor19 polypeptides are useful within genetherapy applications where it is desired to increase or inhibit zcytor19activity. If a mammal has a mutated or absent zcytor19 gene, thezcytor19 gene can be introduced into the cells of the mammal. In oneembodiment, a gene encoding a zcytor19 polypeptide is introduced in vivoin a viral vector. Such vectors include an attenuated or defective DNAvirus, such as, but not limited to, herpes simplex virus (HSV),papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associatedvirus (AAV), and the like. Defective viruses, which entirely or almostentirely lack viral genes, are preferred. A defective virus is notinfective after introduction into a cell. Use of defective viral vectorsallows for administration to cells in a specific, localized area,without concern that the vector can infect other cells. Examples ofparticular vectors include, but are not limited to, a defective herpessimplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci.2:320-30, 1991); an attenuated adenovirus vector, such as the vectordescribed by Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30,1992; and a defective adeno-associated virus vector (Samulski et al., J.Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).

In another embodiment, a zcytor19 gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Kuo et al., Blood 82:845, 1993. Alternatively, the vectorcan be introduced by lipofection in vivo using liposomes. Syntheticcationic lipids can be used to prepare liposomes for in vivotransfection of a gene encoding a marker (Felgner et al., Proc. Natl.Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci.USA 85:8027-31, 1988). The use of lipofection to introduce exogenousgenes into specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. More particularly, directing transfection to particularcells represents one area of benefit. For instance, directingtransfection to particular cell types would be particularly advantageousin a tissue with cellular heterogeneity, such as the pancreas, liver,kidney, and brain. Lipids may be chemically coupled to other moleculesfor the purpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

It is possible to remove the target cells from the body; to introducethe vector as a naked DNA plasmid; and then to re-implant thetransformed cells into the body. Naked DNA vectors for gene therapy canbe introduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Antisense methodology can be used to inhibit zcytor19 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of azcytor19-encoding polynucleotide (e.g., a polynucleotide as set forth inSEQ ID NO:1 SEQ ID NO:18, or SEQ ID NO:20) are designed to bind tozcytor19-encoding mRNA and to inhibit translation of such mRNA. Suchantisense polynucleotides are used to inhibit expression of zcytor19polypeptide-encoding genes in cell culture or in a subject.

In addition, as a cell surface molecule, zcytor19 polypeptides can beused as a target to introduce gene therapy into a cell. This applicationwould be particularly appropriate for introducing therapeutic genes intocells in which zcytor19 is normally expressed, such as lymphoid tissue,bone marrow, prostate, thyroid, and PBLs, or cancer cells which expresszcytor19 polypeptide. For example, viral gene therapy, such as describedabove, can be targeted to specific cell types in which express acellular receptor, such as zcytor19 polypeptide, rather than the viralreceptor. Antibodies, or other molecules that recognize zcytor19molecules on the target cell's surface can be used to direct the virusto infect and administer gene therapeutic material to that target cell.See, Woo, S. L. C, Nature Biotech. 14:1538, 1996; Wickham, T. J. et al,Nature Biotech. 14:1570-1573, 1996; Douglas, J. T et al., NatureBiotech. 14:1574-1578, 1996; Rihova, B., Crit. Rev. Biotechnol.17:149-169, 1997; and Vile, R. G. et al., Mol. Med. Today 4:84-92, 1998.For example, a bispecific antibody containing a virus-neutralizing Fabfragment coupled to a zcytor19-specific antibody can be used to directthe virus to cells expressing the zcytor19 receptor and allow efficiententry of the virus containing a genetic element into the cells. See, forexample, Wickham, T. J., et al., J. Virol. 71:7663-7669, 1997; andWickham, T. J., et al., J. Virol. 70:6831-6838, 1996.

The present invention also provides reagents which will find use indiagnostic applications. For example, the zcytor19 gene, a probecomprising zcytor19 DNA or RNA or a subsequence thereof can be used todetermine if the zcytor19 gene is present on chromosome 1 or if amutation has occurred. Zcytor19 is located at the 1p36.11 region ofchromosome 1. Detectable chromosomal aberrations at the zcytor19 genelocus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Such aberrations can be detected using polynucleotidesof the present invention by employing molecular genetic techniques, suchas restriction fragment length polymorphism (RFLP) analysis,fluorescence in situ hybridization methods, short tandem repeat (STR)analysis employing PCR techniques, and other genetic linkage analysistechniques known in the art (Sambrook et al., ibid.; Ausubel et. al.,ibid.; Marian, Chest 108:255-65, 1995).

The precise knowledge of a gene's position can be useful for a number ofpurposes, including: 1) determining if a sequence is part of an existingcontig and obtaining additional surrounding genetic sequences in variousforms, such as YACs, BACs or cDNA clones; 2) providing a possiblecandidate gene for an inheritable disease which shows linkage to thesame chromosomal region; and 3) cross-referencing model organisms, suchas mouse, which may aid in determining what function a particular genemight have.

The zcytor19 gene is located at the 1p36.11 region of chromosome 1. Oneof skill in the art would recognize that chromosomal aberrations in andaround the 1p36 region are involved in several cancers includingneuroblastoma, melanoma, breast, colon, prostate and other cancers. Suchaberrations include gross chromosomal abnormalities such astranslocations, loss of heterogeneity (LOH) and the like in and around1p36. Thus, a marker in the 1p36.11 locus, such as provided by thepolynucleotides of the present invention, would be useful in detectingtranslocations, aneuploidy, rearrangements, LOH other chromosomalabnormalities involving this chromosomal region that are present incancers. For example, zcytor19 polynucleotide probes can be used todetect abnormalities or genotypes associated with neuroblastoma, whereinLOH between 1p36.1 and 1p36.3 is prevalent, and a breakpoint at 1p36.1is evident. At least 70% of neuroblastomas have cytogenetically visiblechromosomal aberrations in lp, including translocation and deletion, andthat the abnormality is most likely due to complex translocation anddeletion mechanisms. See, for example Ritke, M K et al., Cytogenet. CellGenet. 50:84-90, 1989; and Weith, A et al., Genes Chromosomes Cancer1:159-166, 1989). As zcytor19 is localized to 1p36.11, and fallsdirectly within the region wherin aberrations are prevalent inneuroblastoma, one of skill in the art would apprecitate that thepolynucleotides of the present invention could serve as a diagnostic forneuroblastoma, as well as aid in the elucidation of translocation anddeletion mechanisms that give rise to neuroblastoma. In addition, LOH at1p36 is evident in melanoma (Dracopoli, N C et al, Am. J. Hum. Genet. 45(suppl.):A19, 1989; Dracopoli, N C et al, Proc. Nat. Acad. Sci.86:4614-4618, 1989; Goldstein, A M et al., Am. J. Hum. Genet.52:537-550, 1993); as well as prostate cancer in families with a historyof both prostate and brain cancer (1p36, LOH) (Gibbs, M et al., Am. J.Hum. Genet. 64:776-787, 1999); and breast cancer, wherin deletions andduplications of chromosome 1 are the most common aberrations in breastcarcinoma (1p36) (Kovacs, G. Int. J. Cancer 21:688-694, 1978; Rodgers, Cet al., Cancer Genet. Cytogent. 13:95-119, 1984; and Genuardi, M et al.,Am. J. Hum. Genet. 45:73-82, 1989). Since translocation, LOH and otheraberrations in this region of human chromosome 1 are so prevalent inhuman cancers, and the zcytor19 gene is specifically localized to1p36.11, the polynucleitides of the present invention have use indetecting such aberrations that are clearly associated with humandisease, as deacribed herein.

Moreover, there is further evidence for cancer resulting from mutationsin the 1p36 region wherein zcytor19 is located, and polynucleotideprobes can be used to detect abnormalities or genotypes associatedtherewith: P73, a potential tumor suppressor maps to 1p36 a regionfrequently deleted in neuroblastoma and other cancers (Kaghad, M et al.,Cell 90:809-819, 1997); rhabdomyosarcoma, which involves a translocationat the 1p36.2-p36.12 region of chromosome 1 that results in a fusion ofthe PAX7 gene from chromosome 1 with FKHR gene on choromosome 13;Leukemia-associated Protein (LAP) (1p36.1-p35) is increased in the cellsof various types of leukemia; heparin sulfate proteoglycan (Perlecan)(1p36.1) associated with tumors, and wherein translocations are seen;and colon cancer (1p36-p35). Further, zcytor19 polynucleotide probes canbe used to detect abnormalities or genotypes associated with chromosome1p36.11 deletions and translocations associated with human diseases, andprefereably cancers, as described above. Moreover, amongst other geneticloci, those for Clq complement components (ClQA, B, and G)(1p36.3-p34.1); dyslexia (1p36-p34); lymphoid activation antigen CD30(1p36); sodium channel non-voltage-gated type 1 (1p36.3-p36.2); tumornecrosis factor receptors (TNFRSF1b and TNFRS12) (1p36.3-p36.2) whichlike zcytor19 are cytokine receptors; phospholipase A2 (PLA2) (1p35);rigid spine muscular dystrophy (1p36-p35) all manifest themselves inhuman disease states as well as map to this region of the human genome.See the Online Mendellian Inheritance of Man (OMIM™, National Center forBiotechnology Information, National Library of Medicine. Bethesda, Md.)gene map, and references therein, for this region of human chromosome 1on a publicly available world wide web server. All of these serve aspossible candidate genes for an inheritable disease which show linkageto the same chromosomal region as the zcytor19 gene. Thus, zcytor19polynucleotide probes can be used to detect abnormalities or genotypesassociated with these defects.

Similarly, defects in the zcytor19 gene itself may result in a heritablehuman disease state. The zcytor19 gene (1p36.11) is located near anotherclass II receptor, the zcytor11 cytokine receptor gene (1p35.1)(commonly owned U.S. Pat. No. 5,965,704), as well as TNF receptors(1p36.3-p36.2), suggesting that this chromosomal region is commonlyregulated, and/or important for immune function. Moreover, one of skillin the art would appreciate that defects in cytokine receptors are knownto cause disease states in humans. For example, growth hormone receptormutation results in dwarfism (Amselem, S et al., New Eng. J. Med. 321:989-995, 1989), IL-2 receptor gamma mutation results in severe combinedimmunodeficiency (SCID) (Noguchi, M et al., Cell 73: 147-157, 1993),c-Mpl mutation results in thrombocytopenia (Ihara, K et al., Proc. Nat.Acad. Sci. 96: 3132-3136, 1999), and severe mycobacterial and Salmonellainfections result in interleukin-12 receptor-deficient patients (deJong, R et al., Science 280: 1435-1438, 1998), amongst others. Thus,similarly, defects in zcytor19 can cause a disease state orsusceptibility to disease or infection. As, zcytor19 is a cytokinereceptor in a chromosomal hot spot for aberrations involved in numerouscancers and is shown to be expressed in pre-B-cell acute leukemia cells,and other cancers described herein, the molecules of the presentinvention could also be directly involved in cancer formation ormetastasis. As the zcytor19 gene is located at the 1p36.11 regionzcytor19, polynucleotide probes can be used to detect chromosome 1p36.11loss, trisomy, duplication or translocation associated with humandiseases, such as immune cell cancers, neuroblastoma, bone marrowcancers, thyroid, parathyroid, prostate, melanoma, or other cancers, orimmune diseases. Moreover, molecules of the present invention, such asthe polypeptides, antagonists, agonists, polynucleotides and antibodiesof the present invention would aid in the detection, diagnosisprevention, and treatment associated with a zcytor19 genetic defect.

A diagnostic could assist physicians in determining the type of diseaseand appropriate associated therapy, or assistance in genetic counseling.As such, the inventive anti-zcytor19 antibodies, polynucleotides, andpolypeptides can be used for the detection of zcytor19 polypeptide, mRNAor anti-zcytor19 antibodies, thus serving as markers and be directlyused for detecting or genetic diseases or cancers, as described herein,using methods known in the art and described herein. Further, zcytor19polynucleotide probes can be used to detect abnormalities or genotypesassociated with chromosome 1p36.11 deletions and translocationsassociated with human diseases, other translocations involved withmalignant progression of tumors or other 1p36.11 mutations, which areexpected to be involved in chromosome rearrangements in malignancy; orin other cancers, or in spontaneous abortion. Similarly, zcytor19polynucleotide probes can be used to detect abnormalities or genotypesassociated with chromosome 1p36.11 trisomy and chromosome lossassociated with human diseases. Thus, zcytor19 polynucleotide probes canbe used to detect abnormalities or genotypes associated with thesedefects.

As discussed above, defects in the zcytor19 gene itself may result in aheritable human disease state. Molecules of the present invention, suchas the polypeptides, antagonists, agonists, polynucleotides andantibodies of the present invention would aid in the detection,diagnosis prevention, and treatment associated with a zcytor19 geneticdefect. In addition, zcytor19 polynucleotide probes can be used todetect allelic differences between diseased or non-diseased individualsat the zcytor19 chromosomal locus. As such, the zcytor19 sequences canbe used as diagnostics in forensic DNA profiling.

In general, the diagnostic methods used in genetic linkage analysis, todetect a genetic abnormality or aberration in a patient, are known inthe art. Analytical probes will be generally at least 20 nt in length,although somewhat shorter probes can be used (e.g., 14-17 nt). PCRprimers are at least 5 nt in length, preferably 15 or more, morepreferably 20-30 nt. For gross analysis of genes, or chromosomal DNA, azcytor19 polynucleotide probe may comprise an entire exon or more. Exonsare readily determined by one of skill in the art by comparing zcytor19sequences (SEQ ID NO:1 SEQ ID NO:18, or SEQ ID NO:20) with the humangenomic DNA for zcytor19 (Genbank Accession No. AL358412). In general,the diagnostic methods used in genetic linkage analysis, to detect agenetic abnormality or aberration in a patient, are known in the art.Most diagnostic methods comprise the steps of (a) obtaining a geneticsample from a potentially diseased patient, diseased patient orpotential non-diseased carrier of a recessive disease allele; (b)producing a first reaction product by incubating the genetic sample witha zcytor19 polynucleotide probe wherein the polynucleotide willhybridize to complementary polynucleotide sequence, such as in RFLPanalysis or by incubating the genetic sample with sense and antisenseprimers in a PCR reaction under appropriate PCR reaction conditions;(iii) Visualizing the first reaction product by gel electrophoresisand/or other known method such as visualizing the first reaction productwith a zcytor19 polynucleotide probe wherein the polynucleotide willhybridize to the complementary polynucleotide sequence of the firstreaction; and (iv) comparing the visualized first reaction product to asecond control reaction product of a genetic sample from wild typepatient. A difference between the first reaction product and the controlreaction product is indicative of a genetic abnormality in the diseasedor potentially diseased patient, or the presence of a heterozygousrecessive carrier phenotype for a non-diseased patient, or the presenceof a genetic defect in a tumor from a diseased patient, or the presenceof a genetic abnormality in a fetus or pre-implantation embryo. Forexample, a difference in restriction fragment pattern, length of PCRproducts, length of repetitive sequences at the zcytor19 genetic locus,and the like, are indicative of a genetic abnormality, geneticaberration, or allelic difference in comparison to the normal wild typecontrol. Controls can be from unaffected family members, or unrelatedindividuals, depending on the test and availability of samples. Geneticsamples for use within the present invention include genomic DNA, mRNA,and cDNA isolated form any tissue or other biological sample from apatient, such as but not limited to, blood, saliva, semen, embryoniccells, amniotic fluid, and the like. The polynucleotide probe or primercan be RNA or DNA, and will comprise a portion of SEQ ID NO:1 SEQ IDNO:18, or SEQ ID NO:20 the complement of SEQ ID NO:1, SEQ ID NO:18, orSEQ ID NO:20 or an RNA equivalent thereof. Such methods of showinggenetic linkage analysis to human disease phenotypes are well known inthe art. For reference to PCR based methods in diagnostics see,generally, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), White (ed.), PCR Protocols: Current Methods andApplications (Humana Press, Inc. 1993), Cotter (ed.), MolecularDiagnosis of Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek(eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

Mutations associated with the zcytor19 locus can be detected usingnucleic acid molecules of the present invention by employing standardmethods for direct mutation analysis, such as restriction fragmentlength polymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocolsfor Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)). Direct analysis of an zcytor19 gene for a mutation can beperformed using a subject's genomic DNA. Methods for amplifying genomicDNA, obtained for example from peripheral blood lymphocytes, arewell-known to those of skill in the art (see, for example, Dracopoli etal. (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7(John Wiley & Sons 1998)).

Mice engineered to express the zcytor19 gene, referred to as “transgenicmice,” and mice that exhibit a complete absence of zcytor19 genefunction, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express zcytor19, either ubiquitously or undera tissue-specific or tissue-restricted promoter can be used to askwhether over-expression causes a phenotype. For example, over-expressionof a wild-type zcytor19 polypeptide, polypeptide fragment or a mutantthereof may alter normal cellular processes, resulting in a phenotypethat identifies a tissue in which zcytor19 expression is functionallyrelevant and may indicate a therapeutic target for the zcytor19, itsagonists or antagonists. For example, a preferred transgenic mouse toengineer is one that expresses a “dominant-negative” phenotype, such asone that over-expresses the zcytor19 polypeptide comprising anextracellular cytokine binding domain with the transmembrane domainattached (approximately amino acids 21 (Arg) to 249 (Trp) of SEQ ID NO:2or SEQ ID NO:19; or SEQ ID NO:4 attached in frame to a transmembranedomain). Another preferred transgenic mouse is one that over-expresseszcytor19 soluble receptors, such as those disclosed herein. Moreover,such over-expression may result in a phenotype that shows similaritywith human diseases. Similarly, knockout zcytor19 mice can be used todetermine where zcytor19 is absolutely required in vivo. The phenotypeof knockout mice is predictive of the in vivo effects of a zcytor19antagonist, such as those described herein, may have. The mouse or thehuman zcytor19 cDNA can be used to isolate murine zcytor19 mRNA, cDNAand genomic DNA, which are subsequently used to generate knockout mice.These transgenic and knockout mice may be employed to study the zcytor19gene and the protein encoded thereby in an in vivo system, and can beused as in vivo models for corresponding human or animal diseases (suchas those in commercially viable animal populations). The mouse models ofthe present invention are particularly relevant as tumor models for thestudy of cancer biology and progression. Such models are useful in thedevelopment and efficacy of therapeutic molecules used in human cancers.Because increases in zcytor19 expression, as well as decreases inzcytor19 expression are associated with specific human cancers, bothtransgenic mice and knockout mice would serve as useful animal modelsfor cancer. Moreover, in a preferred embodiment, zcytor19 transgenicmouse can serve as an animal model for specific tumors, particularlyesophagus, liver, ovary, rectum, stomach, and uterus tumors, andmelanoma, B-cell leukemia and other lymphoid cancers. Moreover,transgenic mice expression of zcytor19 antisense polynucleotides orribozymes directed against zcytor19, described herein, can be usedanalogously to transgenic mice described above.

For pharmaceutical use, the soluble receptor polypeptides of the presentinvention are formulated for parenteral, particularly intravenous orsubcutaneous, delivery according to conventional methods. Intravenousadministration will be by bolus injection or infusion over a typicalperiod of one to several hours. In general, pharmaceutical formulationswill include a zcytor19 soluble receptor polypeptide in combination witha pharmaceutically acceptable vehicle, such as saline, buffered saline,5% dextrose in water or the like. Formulations may further include oneor more excipients, preservatives, solubilizers, buffering agents,albumin to prevent protein loss on vial surfaces, etc. Methods offormulation are well known in the art and are disclosed, for example, inRemington: The Science and Practice of Pharmacy, Gennaro, ed., MackPublishing Co., Easton, Pa., 19th ed., 1995. Therapeutic doses willgenerally be in the range of 0.1 to 100 μg/kg of patient weight per day,preferably 0.5-20 mg/kg per day, with the exact dose determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins may be administered for acute treatment, over one week orless, often over a period of one to three days or may be used in chronictreatment, over several months or years. In general, a therapeuticallyeffective amount of zcytor19 soluble receptor polypeptide is an amountsufficient to produce a clinically significant effect.

Polynucleotides and polypeptides of the present invention willadditionally find use as educational tools as a laboratory practicumkits for courses related to genetics and molecular biology, proteinchemistry and antibody production and analysis. Due to its uniquepolynucleotide and polypeptide sequence molecules of zcytor19 can beused as standards or as “unknowns” for testing purposes. For example,zcytor19 polynucleotides can be used as an aid, such as, for example, toteach a student how to prepare expression constructs for bacterial,viral, and/or mammalian expression, including fusion constructs, whereinzcytor19 is the gene to be expressed; for determining the restrictionendonuclease cleavage sites of the polynucleotides; determining mRNA andDNA localization of zcytor19 polynucleotides in tissues (i.e., byNorthern and Southern blotting as well as polymerase chain reaction);and for identifying related polynucleotides and polypeptides by nucleicacid hybridization.

Zcytor19 polypeptides can be used educationally as an aid to teachpreparation of antibodies; identifying proteins by Western blotting;protein purification; determining the weight of expressed zcytor19polypeptides as a ratio to total protein expressed; identifying peptidecleavage sites; coupling amino and carboxyl terminal tags; amino acidsequence analysis, as well as, but not limited to monitoring biologicalactivities of both the native and tagged protein (i.e., receptorbinding, signal transduction, proliferation, and differentiation) invitro and in vivo. Zcytor19 polypeptides can also be used to teachanalytical skills such as mass spectrometry, circular dichroism todetermine conformation, especially of the four alpha helices, x-raycrystallography to determine the three-dimensional structure in atomicdetail, nuclear magnetic resonance spectroscopy to reveal the structureof proteins in solution. For example, a kit containing the zcytor19 canbe given to the student to analyze. Since the amino acid sequence wouldbe known by the professor, the specific protein can be given to thestudent as a test to determine the skills or develop the skills of thestudent, the teacher would then know whether or not the student hascorrectly analyzed the polypeptide. Since every polypeptide is unique,the educational utility of zcytor19 would be unique unto itself.

Moreover, since zcytor19 has a tissue-specific expression and is apolypeptide with a class II cytokine receptor structure and a distinctchromosomal localization, and expressin pattern, activity can bemeasured using proliferation assays; luciferase and binding assaysdescribed herein. Moreover, expression of zcytor19 polynucleotides andpolypeptides in lymphoid and other tissues can be analyzed in order totrain students in the use of diagnostic and tissue-specificidentification and methods. Moreover zcytor19 polynucleotides can beused to train students on the use of chromosomal detection anddiagnostic methods, since it's locus is known. Moreover, students can bespecifically trained and educated about human chromosome 1, and morespecifically the locus 1p36.11 wherein the zcytor19 gene is localized.Such assays are well known in the art, and can be used in an educationalsetting to teach students about cytokine receptor proteins and examinedifferent properties, such as cellular effects on cells, enzymekinetics, varying antibody binding affinities, tissue specificity, andthe like, between zcytor19 and other cytokine receptor polypeptides inthe art.

The antibodies which bind specifically to zcytor19 can be used as ateaching aid to instruct students how to prepare affinity chromatographycolumns to purify zcytor19, cloning and sequencing the polynucleotidethat encodes an antibody and thus as a practicum for teaching a studenthow to design humanized antibodies. Moreover, antibodies which bindspecifically to zcytor19 can be used as a teaching aid for use indetection of B-cell tumor tissue, esophagus, liver, ovary, rectum,stomach, and uterus tumors, and melanoma, pre-B-cell lymphoblasticleukemia and other lymphoid cancers using histological, and in situmethods amongst others known in the art. The zcytor19 gene, polypeptideor antibody would then be packaged by reagent companies and sold touniversities and other educational entities so that the students gainskill in art of molecular biology. Because each gene and protein isunique, each gene and protein creates unique challenges and learningexperiences for students in a lab practicum. Such educational kitscontaining the zcytor19 gene, polypeptide or antibody are consideredwithin the scope of the present invention.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Identification and Isolation of Full-length HumanZcytor19 cDNA

Zcytor19 was identified as a predicted full-length cDNA from humangenomic DNA AL358412 (Genbank). The sequence of the predicted fulllength zcytor19 polynucleotide is shown in SEQ ID NO:1 and thecorresponding polypeptide is shown in SEQ ID NO:2. A variant full-lengthzcytor19 cDNA sequence was identified and is shown in SEQ ID NO:18 andthe corresponding polynucleotides shown in SEQ ID NO:19. Moreover, atruncated soluble form of zcytor19 cDNA sequence was identified and isshown in SEQ ID NO:20 and the corresponding polypeptide is shown in SEQID NO:21.

Example 2 Tissue Distribution in Tissue Panels Using Northern Blot andPCR

A. Human Zcytor19 Tissue Distribution Using Northern Blot

Human Multiple Tissue Northern Blots (Human 12-lane MTN Blot I and II,and Human Immune System MTN Blot II) (Clontech) are probed to determinethe tissue distribution of human zcytor19 expression. A PCR derivedprobe that hybridizes to SEQ ID NO:1 or SEQ ID NO:18 is amplified usingstandard PCR amplification methods. An exemplary PCR reaction is carriedout as follows using primers designed to hybridize to SEQ ID NO:1, SEQID NO:18 or its complement: 30 cycles of 94° C. for 1 minute, 65° C. for1 minute, and 72° C. for 1 minute; followed by 1 cycle at 72° C. for 7minutes. The PCR product is visualized by agarose gel electrophoresisand the PCR product is gel purified as described herein. The probe isradioactively labeled using, e.g., the PRIME IT II™ Random PrimerLabeling Kit (Stratagene) according to the manufacturer's instructions.The probe is purified using, e.g., a NUCTRAP™ push column (Stratagene).EXPRESSHYB™ (Clontech) solution is used for the prehybridization and asa hybridizing solution for the Northern blots. Prehybridization iscarried out, for example, at 68° C. for 2 hours. Hybridization takesplace overnight at about 68° C. with about 1.5×10⁶ cpm/ml of labeledprobe. The blots are washed three times at room temperature in 2×SSC,0.05% SDS, followed by 1 wash for 10 minutes in 2×SSC, 0.1% SDS at 50°C. After exposure to X-ray film, a transcript corresponding to thelength of SEQ ID NO:1 SEQ ID NO:18, or SEQ ID NO:20 or of an mRNAencoding SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 is expected to beseen in tissues that specifically express zcytor19, but not othertissues.

Northern analysis is also performed using Human Cancer Cell Line MTN™(Clontech). PCR and probing conditions are as described above. A strongsignal in a cancer line suggests that zcytor19 expression may beexpressed in activated cells and/or may indicate a cancerous diseasestate. Moreover, using methods known in the art, Northern blots or PCRanalysis of activated lymphocyte cells can also show whether zcytor19 isexpressed in activated immune cells. Based on electronic Northerninformation zcytor19 was shown to be expressed specifically in pre-Bcell acute lymphoblastic leukemia cells.

B. Tissue Distribution in Tissue Panels Using PCR

A panel of cDNAs from human tissues was screened for zcytor19 expressionusing PCR. The panel was made in-house and contained 94 marathon cDNAand cDNA samples from various normal and cancerous human tissues andcell lines are shown in Table 5, below. The cDNAs came from in-houselibraries or marathon cDNAs from in-house RNA preps, Clontech RNA, orInvitrogen RNA. The marathon cDNAs were made using the marathon-Ready™kit (Clontech, Palo Alto, Calif.) and QC tested with clathrin primersZC21195 (SEQ ID NO:6) and ZC21196 (SEQ ID NO:7) and then diluted basedon the intensity of the clathrin band. To assure quality of the panelsamples, three tests for quality control (QC) were run: (1) To assessthe RNA quality used for the libraries, the in-house cDNAs were testedfor average insert size by PCR with vector oligos that were specific forthe vector sequences for an individual cDNA library; (2) Standardizationof the concentration of the cDNA in panel samples was achieved usingstandard PCR methods to amplify full length alpha tubulin or G3PDH cDNAusing a 5′ vector oligo ZC14,063 (SEQ ID NO:8) and 3′ alpha tubulinspecific oligo primer ZC17,574 (SEQ ID NO:9) or 3′ G3PDH specific oligoprimer ZC17,600 (SEQ ID NO:10); and (3) a sample was sent to sequencingto check for possible ribosomal or mitochondrial DNA contamination. Thepanel was set up in a 96-well format that included a human genomic DNA(Clontech, Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA. The PCR was set up usingoligos ZC37685 (SEQ ID NO:26) and ZC37681 (SEQ ID NO:27), TaKaRa Ex Taq™(TAKARA Shuzo Co LTD, Biomedicals Group, Japan), and Rediload dye(Research Genetics, Inc., Huntsville, Ala.). The amplification wascarried out as follows: 1 cycle at 94° C. for 2 minutes, 5 cycles of 94°C. for 30 seconds, 70° C. for 30 seconds, 35 cycles of 94° C. for 30seconds, 64° C. for 30 seconds and 72° C. for 30 seconds, followed by 1cycle at 72° C. for 5 minutes. About 10 μl of the PCR reaction productwas subjected to standard Agarose gel electrophoresis using a 4% agarosegel. The correct predicted DNA fragment size was observed in adrenalgland, bladder, cervix, colon, fetal heart, fetal skin, liver, lung,melanoma, ovary, salivary gland, small intestine, stomach, brain, fetalliver, kidney, prostate, spinal cord, thyroid, placenta, testis, tumoresophagus, tumor liver, tumor ovary, tumor rectum, tumor stomach, tumoruterus, bone marrow, CD3+ library, HaCAT library, HPV library and HPVSlibrary. As this primer pair does not span an intron, there may be riskthat some tissues that are contaminated with genomic DNA or unprocessedmRNA messages would create a false positive in this assay.

Therefore, a different primer pair ZC38481 (SEQ ID NO:47) and ZC38626(SEQ ID NO:48) that span introns were used using the methods describedabove, to re-evaluate the tissue distribution. The correct predicted DNAfragment size (256 bp) was observed in colon, fetal heart, fetal liver,kidney, liver, lung, mammary gland, prostate, salivary gland, smallintestine, adipocyte library, brain library, islet library, and prostatelibrary, RPMI 1788 (B-cell line), spinal cord, placenta library, testis,tumor esophagus, tumor ovary, tumor rectum, tumor stomach, HaCATlibrary, HPV library and HPVS library.

Mouse tissue panels were also examined using another set of primerpairs: (1) ZC38706 (SEQ ID NO:49) and ZC38711 (SEQ ID NO:50) (800 bpproduct) using the methods described above. This panel showed a limitedtissue distribution for mouse zcytor19: mouse prostate cell lines,salivary gland library, and skin.

TABLE 5 Tissue/Cell line #samples Adrenal gland 1 Bladder 1 Bone Marrow1 Brain 1 Cervix 1 Colon 1 Fetal brain 1 Fetal heart 1 Fetal kidney 1Fetal liver 1 Fetal lung 1 Fetal muscle 1 Fetal skin 1 Heart 2 K562(ATCC # CCL-243) 1 Kidney 1 Liver 1 Lung 1 Lymph node 1 Melanoma 1Pancreas 1 Pituitary 1 Placenta 1 Prostate 1 Rectum 1 Salivary Gland 1Skeletal muscle 1 Small intestine 1 Spinal cord 1 Spleen 1 Stomach 1Testis 2 Thymus 1 Thyroid 1 Trachea 1 Uterus 1 Esophagus tumor 1 Gastrictumor 1 Kidney tumor 1 Liver tumor 1 Lung tumor 1 Ovarian tumor 1 Rectaltumor 1 Uterus tumor 1 Bone Marrow 3 Fetal brain 3 Islet 2 Prostate 3RPMI #1788 (ATCC # CCL-156) 2 Testis 4 Thyroid 2 WI38 (ATCC # CCL-75 2ARIP (ATCC # CRL-1674 - rat) 1 HaCat - human keratinocytes 1 HPV (ATCC #CRL-2221) 1 Adrenal gland 1 Prostate SM 2 CD3+ selected PBMC's 1Ionomycin + PMA stimulated HPVS (ATCC # CRL-2221) - selected 1 Heart 1Pituitary 1 Placenta 2 Salivary gland 1 HL60 (ATCC # CCL-240) 3 Platelet1 HBL-100 1 Renal mesangial 1 T-cell 1 Neutrophil 1 MPC 1 Hut-102 (ATCC# TIB-162) 1 Endothelial 1 HepG2 (ATCC # HB-8065) 1 Fibroblast 1 E.Histo 1

Example 3 PCR-Based Chromosomal Mapping of the Zcytor19 Gene

Zcytor19 is mapped to chromosome 1 using the commercially available“GeneBridge 5 4 Radiation Hybrid (RH) Mapping Panel” (Research Genetics,Inc., Huntsville, Ala.). The GeneBridge 4 RH panel contains DNA fromeach of 93 radiation hybrid clones, plus two control DNAs (the HFL donorand the A23 recipient). A publicly available WWW server allows mappingrelative to the Whitehead Institute/MIT Center for Genome Research'sradiation hybrid map of the human genome (the “WICGR” radiation hybridmap) which is 10 constructed with the GeneBridge 4 RH panel.

For the mapping of Zcytor19 with the GeneBridge 4 RH panel, 20 μlreactions are set up in a 96-well microtiter plate compatible for PCR(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient96”thermal cycler (Stratagene). Each of the 95 PCR reactions consistedof 2 μl 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc.,Palo Alto, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, FosterCity, Calif.), 1 μl sense primer, ZC27,895 (SEQ ID NO:14), 1 μlantisense primer, ZC27,899 (SEQ ID NO:24), 2 μl “RediLoad” (ResearchGenetics, Inc., Huntsville, Ala.), 0.4 μl 50× Advantage KlenTaqPolymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from anindividual hybrid clone or control and distilled water for a totalvolume of 20 μl. The reactions are overlaid with an equal amount ofmineral oil and sealed. The PCR cycler conditions are as follows: aninitial 1 cycle 5 minute denaturation at 94° C., 35 cycles of a 45seconds denaturation at 94° C., 45 seconds annealing at 54° C. and 1minute AND 15 seconds extension at 72° C., followed by a final 1 cycleextension of 7 minutes at 72° C. The reactions are separated byelectrophoresis on a 2% agarose gel (EM Science, Gibbstown, N.J.) andvisualized by staining with ethidium bromide. The results show thatZcytor19 maps on the chromosome 1 WICGR radiation hybrid map in the1p36.11 chromosomal region.

Example 4 Construction of Mammalian Expression Vectors that ExpressZcytor19 Soluble Receptors: Zcytor19CEE, Zcytor19CFLG zcytor19CHIS andZcytor19-Fc4

A. Construction of Zcytor19 Mammalian Expression Vector ContainingZcytor19CEE, Zcytor19CFLG and Zcytor19CHIS

An expression vector is prepared for the expression of the soluble,extracellular domain of the zcytor19 polypeptide, pC4zcytor19CEE,wherein the construct is designed to express a zcytor19 polypeptidecomprised of the predicted initiating methionine and truncated adjacentto the predicted transmembrane domain, and with a C-terminal Glu-Glu tag(SEQ ID NO:11).

A zcytor19 DNA fragment comprising the zcytor19 extracellular orcytokine binding domain of zcytor19 described herein, is created usingPCR, and purified using standard methods. The excised DNA is subclonedinto a plasmid expression vector that has a signal peptide, e.g., thenative zcytor19 signal peptide, and attaches a Glu-Glu tag (SEQ IDNO:11) to the C-terminus of the zcytor19 polypeptide-encodingpolynucleotide sequence. Such a mammalian expression vector contains anexpression cassette having a mammalian promoter, multiple restrictionsites for insertion of coding sequences, a stop codon and a mammalianterminator. The plasmid can also have an E. coli origin of replication,a mammalian selectable marker expression unit having an SV40 promoter,enhancer and origin of replication, a DHFR gene and the SV40 terminator.

Restriction digested zcytor19 insert and previously digested vector areligated using standard molecular biological techniques, andelectroporated into competent cells such as DH10B competent cells (GIBCOBRL, Gaithersburg, Md.) according to manufacturer's direction and platedonto LB plates containing 50 mg/ml ampicillin, and incubated overnight.Colonies are screened by restriction analysis of DNA prepared fromindividual colonies. The insert sequence of positive clones is verifiedby sequence analysis. A large scale plasmid preparation is done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

The same process is used to prepare the zcytor19 soluble receptors witha C-terminal his tag, composed of 6 His residues in a row; and aC-terminal FLAG® tag (SEQ ID NO:12), zcytor19CFLAG. To construct theseconstructs, the aforementioned vector has either the CHIS or the FLAG®tag in place of the glu-glu tag (SEQ ID NO:11).

B. Mammalian Expression Construction of Soluble Human Zcytor19 Receptor:Zcytor19-Fc4

An expression vector, zcytor19/Fc4/pzmp20, was prepared to express aC-terminally Fc4 tagged soluble version of zcytor19 (human zcytor19-Fc4)in BHK cells. A fragment of zcytor19 cDNA that includes thepolynucleotide sequence from extracellular domain of the zcytor19receptor was fused in frame to the Fc4 polynucleotide sequence (SEQ IDNO:13) to generate a zcytor19-Fc4 fusion (SEQ ID NO:22 and SEQ IDNO:23). The pzmp20 vector is a mammalian expression vector that containsthe Fc4 polynucleotide sequence and a cloning site that allows rapidconstruction of C-terminal Fc4 fusions using standard molecular biologytechniques.

A 630 base pair fragment was generated by PCR, containing theextracellular domain of human zcytor19 with BamHI and Bg12 sites codedon the 5′ and 3′ ends, respectively. This PCR fragment was generatedusing primers ZC37967 (SEQ ID NO:24) and ZC37972 (SEQ ID NO:25) byamplification from human brain cDNA library. The PCR reaction conditionswere as follows: 30 cycles of 94° C. for 20 seconds, and 68° C. for 2minutes; 1 cycle at 68° C. for 4 minutes; followed by a 10° C. soak. Thefragment was digested with BamHI and Bg12 restriction endonucleases andsubsequently purified by 1% gel electrophoresis and band purificationusing QiaQuick gel extraction kit (Qiagen). The resulting purified DNAwas ligated for 5 hours at room temperature into a pzmp20 vectorpreviously digested with Bg12 containing Fc4 3′ of the Bg12 sites.

One μl of the ligation mix was electroporated in 37 μl DHIOBelectrocompetent E. coli (Gibco) according to the manufacturer'sdirections. The transformed cells were diluted in 400 μl of LB media andplated onto LB plates containing 100 μg/ml ampicillin. Clones wereanalyzed by restriction digests and positive clones were sent for DNAsequencing to confirm the sequence of the fusion construct.

Example 5 Transfection and Expression of Zcytorl 9 Soluble ReceptorPolypeptides

A. Mammalian Expression Human Zcytor19 Soluble Receptor: Zcytor19/Fc4

BHK 570 cells (ATCC NO:CRL-10314) were plated in T-75 tissue cultureflasks and allowed to grow to approximately 50 to 70% confluence at 37°C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose, (Gibco BRL,Gaithersburg, Md.), 5% fetal bovine serum, 1 mM L-glutamine (JRHBiosciences, Lenea, Kans.), 1 mM sodium pyruvate (Gibco BRL)). The cellswere then transfected with the plasmid zcytor19/Fc4/pzmp20 (Example 4B)using Lipofectamine™ (Gibco BRL), in serum free (SF) media formulation(DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1%L-glutamine and 1% sodium pyruvate). Ten μg of the plasmid DNA zcytorl9/Fc4/pzmp20 (Example 4B) was diluted into a 15ml tube to a total finalvolume of 500 μl with SF media. 50 μl of Lipofectamine was mixed with450 μl of SF medium. The Lipofectamine mix was added to the DNA mix andallowed to incubate approximately 30 minutes at room temperature. Fourml of SF media was added to the DNA:Lipofectamine mixture. The cellswere rinsed once with 5 ml of SF media, aspirated, and theDNA:Lipofectamine mixture was added. The cells were incubated at 37° C.for five hours, and then 5 ml of DMEM/10%FBS media was added. The flaskwas incubated at 37° C. overnight after which time the cells were splitinto the selection media (DMEM/FBS media from above with the addition of1 μM methotrexate (Sigma Chemical Co., St. Louis, Mo.) in 150 mm platesat 1:2, 1:10, and 1:50. Approximately 10 days post-transfection, one 150mm plate of 1 μM methotrexate resistant colonies was trypsinized, thecells were pooled, and one-half of the cells were replated in 10 μMmethotrexate; to further amplify expression of the zcytor19/Fc4 protein.A conditioned-media sample from this pool of amplified cells was testedfor expression levels using SDS-PAGE and Western analysis.

Single clones expressing the soluble receptors can also isolated,screened and grown up in cell culture media, and purified using standardtechniques. Moreover, CHO cells are also suitable cells for suchpurposes.

Example 6 Assessing Zcytor19 Receptor Heterodimerization Using ORIGENAssay

Soluble zcytor19 receptor zcytor19CFLAG (Example 4 and Example 5), orgp130 (Hibi, M. et al., Cell 63:1149-1157, 1990) are biotinylated byreaction with a five-fold molar excess of sulfo-NHS-LC-Biotin (Pierce,Inc., Rockford, Ill.) according to the manufacturer's protocol. Solublezcytor19 receptor and another soluble receptor subunit, for example,soluble class II cytokine receptors, for example, interferon-gamma,alpha and beta chains and the interferon-alpha/beta receptor alpha andbeta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4,DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) solublereceptors. Receptors in this subfamily may associate to form homodimersthat transduce a signal. These soluble receptors are labeled with a fivefold molar excess of Ru-BPY-NHS (Igen, Inc., Gaithersburg, Md.)according to manufacturer's protocol. The biotinylated andRu-BPY-NHS-labeled forms of the soluble zcytor19 receptor can berespectively designated Bio-zcytor19 receptor and Ru-zcytor19; thebiotinylated and Ru-BPY-NHS-labeled forms of the other soluble receptorsubunit can be similarly designated. Assays can be carried out usingconditioned media from cells expressing a ligand that binds zcytor19heterodimeric receptors, or using purified ligands. Preferred ligandsare those that can bind class II heterodimeric cytokine receptors suchas, IL-10, IL-9, IL-TIF, interferons, TSLP (Levine, S D et al., ibid.;Isaksen, D E et al., ibid.; Ray, R J et al., ibid.; Friend, S L et al.,ibid.), and the like.

For initial receptor binding characterization a panel of cytokines orconditioned medium are tested to determine whether they can mediatehomodimerization of zcytor19 receptor and if they can mediate theheterodimerization of zcytor19 receptor with the soluble receptorsubunits described above. To do this, 50 μl of conditioned media orTBS-B containing purified cytokine, is combined with 50 μl of TBS-B (20mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2) containing e.g., 400 ng/ml ofRu-zcytor19 receptor and Bio-zcytor19, or 400 ng/ml of Ru-zcytor19receptor and e.g., Bio-gp130, or 400 ng/ml of e.g., Ru-classllsubunitand Bio-zcytor19. Following incubation for one hour at room temperature,30 μg of streptavidin coated, 2.8 mm magnetic beads (Dynal, Inc., Oslo,Norway) are added and the reaction incubated an additional hour at roomtemperature. 200 μl ORIGEN assay buffer (Igen, Inc., Gaithersburg, Md.)is then added and the extent of receptor association measured using anM8 ORIGEN analyzer (Igen, Inc.).

Example 7 Construct for Generating a Zcytor19 Receptor Heterodimer

A vector expressing a secreted human zcytor19 heterodimer isconstructed. In this construct, the extracellular cytokine-bindingdomain of zcytor19 is fused to the heavy chain of IgG gamma 1 (IgGγ1)(SEQ ID NO:14 and SEQ ID NO:15), while the extracellular portion of theheteromeric cytokine receptor subunit (E.g., class II cytokinereceptors, for example, interferon-gamma, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonlyowned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly ownedU.S. Pat. No. 5,945,511) receptors)) is fused to a human kappa lightchain (human κ light chain) (SEQ ID NO:16 and SEQ ID NO:17).

A. Construction of IgG Gamma 1 and Human κ Light Chain Fusion Vectors

The heavy chain of IgGγ1 (SEQ ID NO:14) is cloned into the Zem229Rmammalian expression vector (ATCC deposit No. 69447) such that anydesired cytokine receptor extracellular domain having a 5′ EcoRI and 3′NheI site can be cloned in resulting in an N-terminal extracellulardomain-C-terminal IgGγ1 fusion. The IgGγ1 fragment used in thisconstruct is made by using PCR to isolate the IgGγ1 sequence from aClontech hFetal Liver cDNA library as a template. PCR products arepurified using methods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with oligos thatcomprise an MluI/EcoRI linker, into Zem229R previously digested with andEcoRI using standard molecular biology techniques disclosed herein.

The human κ light chain (SEQ ID NO:16) is cloned in the Zem228Rmammalian expression vector (ATCC deposit No. 69446) such that anydesired cytokine receptor extracellular domain having a 5′ EcoRI siteand a 3′ KpnI site can be cloned in resulting in a N-terminal cytokineextracellular domain-C-terminal human κ light chain fusion. As a KpnIsite is located within the human κ light chain sequence (cleaved by theKpnI enzyme after nucleotide 62 in SEQ ID NO:16), a special primer isdesigned to clone the 3′ end of the desired extracellular domain of acytokine receptor into this KpnI site: The primer is designed so thatthe resulting PCR product contains the desired cytokine receptorextracellular domain with a segment of the human κ light chain up to theKpnI site (SEQ ID NO:16). This primer preferably comprises a portion ofat least 10 nucleotides of the 3+ end of the desired cytokine receptorextracellular domain fused in frame 5′ to SEQ ID NO: 16. The human κlight chain fragment used in this construct is made by using PCR toisolate the human κ light chain sequence from the same Clontech humanFetal Liver cDNA library used above. PCR products are purified usingmethods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with theMluI/EcoRI linker described above, into Zem228R previously digested withand EcoRI using standard molecular biology techniques disclosed herein.

B. Insertion of Zcytor19 Receptor or Heterodimeric Subunit ExtracellularDomains into Fusion Vector Constructs

Using the construction vectors above, a construct having zcytor19 fusedto IgGγ1 is made. This construction is done by PCRing the extracellulardomain or cytokine-binding domain of zcytor19 receptor described hereinfrom a prostate cDNA library (Clontech) or activated lymphocyte cDNAlibrary using standard methods, and oligos that provide EcoRI and NheIrestriction sites. The resulting PCR product is digested with EcoRI andNheI, gel purified, as described herein, and ligated into a previouslyEcoRI and NheI digested and band-purified Zem229R/IgGγ1 described above.The resulting vector is sequenced to confirm that the zcytor19/IgG gamma1 fusion (zcytor19/Chl IgG) is correct.

A separate construct having a heterodimeric cytokine receptor subunitextracellular domain fused to κ light is also constructed as above. Thecytokine receptor/human κ light chain construction is performed as aboveby PCRing from, e.g., a lymphocyte cDNA library (Clontech) usingstandard methods, and oligos that provide EcoRI and KpnI restrictionsites. The resulting PCR product is digested with EcoRI and KpnI andthen ligating this product into a previously EcoRI and KpnI digested andband-purified Zem228R/human κ light chain vector described above. Theresulting vector is sequenced to confirm that the cytokine receptorsubunit/human κ light chain fusion is correct.

D. Co-expression of the Zcytor19 and Heterodimeric Cytokine ReceptorSubunit Extracellular Domain

Approximately 15 μg of each of vectors above, are co-transfected intomammalian cells, e.g., BHK-570 cells (ATCC No. CRL-10314) usingLipofectaminePlus™ reagent (Gibco/BRL), as per manufacturer'sinstructions. The transfected cells are selected for 10 days in DMEM+5%FBS (Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St. Louis,Mo.) and 0.5 mg/ml G418 (Gibco/BRL) for 10 days. The resulting pool oftransfectants is selected again in 10 pm of MTX and 0.5 mg/ml G418 for10 days.

The resulting pool of doubly selected cells is used to generate protein.Three Factories (Nunc, Denmark) of this pool are used to generate 10 Lof serum free conditioned medium. This conditioned media is passed overa 1 ml protein-A column and eluted in about 10, 750 microliterfractions. The fractions having the highest protein concentration arepooled and dialyzed (10 kD MW cutoff) against PBS. Finally the dialyzedmaterial is submitted for amino acid analysis (AAA) using routinemethods.

Example 8 Reconstitution of Zcytor19 Receptor In Vitro

To identify components involved in the zcytor19-signaling complex,receptor reconstitution studies are performed as follows. For example,BHK 570 cells (ATCC No. CRL-10314) transfected, using standard methodsdescribed herein, with a luciferase reporter mammalian expression vectorplasmid serve as a bioassay cell line to measure signal transductionresponse from a transfected zcytor19 receptor complex to the luciferasereporter in the presence of zcytor19 Ligand. BHK cells would be used inthe event that BHK cells do not endogenously express the zcytor19receptor. Other cell lines can be used. An exemplary luciferase reportermammalian expression vector is the KZ134 plasmid which is constructedwith complementary oligonucleotides that contain STAT transcriptionfactor binding elements from 4 genes. A modified c-fos Sis inducibleelement (m67SIE, or hSIE) (Sadowski, H. et al., Science 261:1739-1744,1993), the p21 SIE1 from the p21 WAFI gene (Chin, Y. et al., Science272:719-722, 1996), the mammary gland response element of the β-caseingene (Schmitt-Ney, M. et al., Mol. Cell. Biol. 11:3745-3755, 1991), anda STAT inducible element of the Fcg RI gene, (Seidel, H. et al., Proc.Natl. Acad. Sci. 92:3041-3045, 1995). These oligonucleotides containAsp718-XhoI compatible ends and are ligated, using standard methods,into a recipient firefly luciferase reporter vector with a c-Fospromoter (Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998)digested with the same enzymes and containing a neomycin selectablemarker. The KZ134 plasmid is used to stably transfect BHK, or BaF3cells, using standard transfection and selection methods, to make aBHK/KZ134 or BaF3/KZ134 cell line respectively.

The bioassay cell line is transfected with zcytor19 receptor alone, orco-transfected with zcytor19 receptor along with one of a variety ofother known receptor subunits. Receptor complexes include but are notlimited to zcytor19 receptor only, various combinations of zcytor19receptor with class II cytokine receptors, for example,interferon-gamma, alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No.5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No.5,945,511) receptors. Each independent receptor complex cell line isthen assayed in the presence of cytokine-conditioned media or purifiedcytokines and luciferase activity measured using routine methods. Theuntransfected bioassay cell line serves as a control for the backgroundluciferase activity, and is thus used as a baseline to compare signalingby the various receptor complex combinations. The conditioned medium orcytokine that binds the zcytor19 receptor in the presence of the correctreceptor complex, is expected to give a luciferase readout ofapproximately 5 fold over background or greater.

As an alternative, a similar assay can be performed wherein the aBaf3/zcytor19 cell line isco-transfected as described herein andproliferation is measured, using a known assay such as a standard AlamarBlue proliferation assay.

Example 9 COS Cell Transfection and Secretion Trap

A secretion trap assay can be used to identify the zcytor19 receptorligand. Since zcytor19 is a Class II cytokine receptor, the binding ofzcytor19sR/Fc4 fusion protein with known or orphan cytokines was tested.The pZP7 expression vectors containing cDNAs of cytokines (includinghuman IL-TIF, interferon alpha, interferon beta, interferon gamma,IL-10, amongst others) are transfected into COS cells, and the bindingof zcytor19sR/Fc4 to transfected COS cells are carried out using thesecretion trap assay described below. Positive binding in this assayshows potential zcytor19 receptor-ligand pairs.

A. COS Cell Transfections

The COS cell transfection was performed as follows: Mix 0.75 □g cytokineDNA in 50 μl serum free DMEM media (55 mg sodium pyruvate, 146 mgL-glutamine, 5 mg transferrin, 2.5 mg insulin, 1 μg selenium and 5 mgfetuin in 500 ml DMEM (Gibco BRL)), with 5 μl Lipofectamine™ and 45 μlserum free DMEM media. Incubate at room temperature for 30 minutes andthen add 400 μl serum free DMEM media. Add this 500 μl mixture onto1.5×10⁵ COS cells/well plated on 12-well tissue culture plate andincubate for 5 hours at 37° C. Add 500 μl 20% FBS DMEM media (100 mlFBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml DMEM) andincubate overnight.

B. Secretion Trap Assay

The secretion trap was performed as follows: Media was rinsed off cellswith PBS and then fixed for 15 minutes with 1.8% Formaldehyde in PBS.Cells were then washed 2 times with TNT (0.1M Tris-HCL, 0.15M NaCl, and0.05% Tween-20 in H₂O), and permeabilized with 0.1% Triton-X in PBS for15 minutes, and washed 3 times with TNT. Cells were blocked for 1 hourwith TNB (0.1M Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NENRenaissance TSA-Direct Kit) in H₂O. The cells were incubated for 1 hourwith 1 μg/ml, 0.5 μg/ml, or 0.25 μg/ml zcytor19-Fc4 soluble receptorfusion protein (Example 10) in TNB. Cells were then washed 3 times withTNT and were incubated for another hour with 1:1000 dilutedgoat-anti-human Ig-HRP (Fcγ y specific) (Jackson Immuno Research) inTNB. Again cells were washed with TNT.

Positive binding was detected with fluorescein tyramide reagent diluted1:50 in dilution buffer (NEN kit) and incubated for 4.5 minutes, andwashed with TNT. Cells were preserved with Vectashield Mounting Media(Vector Labs Burlingame, Calif.) diluted 1:5 in TNT. Cells werevisualized using a FITC filter on fluorescent microscope.

Since zcytor19 is a Class II cytokine receptor, the binding ofzcytor19sR/Fc4 fusion protein with known or orphan cytokines is tested.The pZP7 expression vectors containing cDNAs of cytokines (includinghuman IL-TIF, interferon alpha, interferon beta, interferon gamma,IL-10, amongst others are transfected into COS cells, and the binding ofzcytor19sR/Fc4 to transfected COS cells are carried out using thesecretion trap assay described above. Positive binding in this assayshows potential zcytor19 receptor-ligand pairs.

Example 10 Expression of Human Zcytor19 in E. coli

A. Construction of Zcytor19-MBP Fusion Expression VectorpTAP170/Zcytor19

An expression plasmid containing a polynucleotide encoding part of thehuman zcytor19 fused N-terminally to maltose binding protein (MBP) wasconstructed via homologous recombination. A fragment of human zcytor19cDNA (SEQ ID NO:1) was isolated using PCR. Two primers were used in theproduction of the human zcytor19 fragment in a PCR reaction: (1) PrimerZC39204 (SEQ ID NO:30), containing 40 bp of the vector flanking sequenceand 24 bp corresponding to the amino terminus of the human zcytor19, and(2) primer ZC39205 (SEQ ID NO:31), containing 40 bp of the 3′ endcorresponding to the flanking vector sequence and 24 bp corresponding tothe carboxyl terminus of the human zcytor19. The PCR reaction conditionswere as follows: 1 cycle of 94C. for 1 minute. Then 20 cycles of 94° C.for 30 seconds, 60° C. for 30 seconds, and 68° C. for 1.5 minutes;followed by 4° C. soak, run in duplicate. Five μl of each 100 μl PCRreaction were run on a 1.0% agarose gel with 1×TBE buffer for analysis,and the expected band of approximately 700 bp fragment was seen. Theremaining 95 μl of PCR reaction was combined with the second PCR tubeprecipitated with 400 μl of absolute ethanol and resuspended in 10 μl ofwater to be used for recombining into the Smal cut recipient vectorpTAP170 to produce the construct encoding the MBP-human zcytor19 fusion,as described below.

Plasmid pTAP170 was derived from the plasmids pRS316 and pMAL-c2. Theplasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P.and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E. coliexpression plasmid. It carries the tac promoter driving Ma1E (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP170 was constructed usingyeast homologous recombination. 100 ng of EcoRl cut pMAL-c2 wasrecombined with 1 μg Pvul cut pRS316, 1 μg linker, and 1 μg Scal/EcoRlcut pRS316. The linker consisted of oligos zc19,372 (100 pmole):zc19,351 (1 pmole): zc19,352 (1 pmole), and zc19,371 (100 pmole)combined in a PCR reaction. Conditions were as follows: 10 cycles of 94°C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 30 seconds;followed by 4° C. soak. PCR products were concentrated via 100% ethanolprecipitation.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl of a mixture containing approximately 1 μg of thehuman zcytor19 insert, and 100 ng of Smal digested pTAP170 vector, andtransferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixturewas electropulsed at 0.75 kV (5 kV/cm), infinite ohms, 25 μF. To eachcuvette was added 600 μl of 1.2 M sorbitol. The yeast was then plated intwo 300 μl aliquots onto two -URA D plates and incubated at 30° C.

After about 48 hours, the Ura+ yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 1 ml of lysis buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 500 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

Transformation of electrocompetent E. coli cells (MC1061, Casadaban et.al. J. Mol. Biol. 138, 179-207) was done with 1 μl yeast DNA prep and 40μl of MC1061 cells. The cells were electropulsed at 2.0 kV, 25 μF and400 ohms. Following electroporation, 0.6 ml SOC (2% BactoÎ Tryptone(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mMKCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added to the cells.After incubation for one hour at 37° C., the cells were plated in onealiquot on LB Kan plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco),30 mg/L kanamycin).

Individual clones harboring the correct expression construct for humanzcytor19 were identified by expression. Cells were grown in SuperbrothII (Becton Dickinson) with 30 μg/ml of kanamycin ovemight. 50 μl of theovernight culture was used to inoculate 2 ml of fresh Superbroth II +30μg/ml kanamycin. Cultures were grown at 37° C., shaking for 2 hours. 1ml of the culture was induced with 1 mM IPTG. 2-4 hours later the 250 μlof each culture was mixed 250 μl Thomer buffer with 5% βME and dye (8Murea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples wereboiled for 5-10 minutes. 20 μl were loaded per lane on a 4%-12% PAGE gel(NOVEX). Gels were run in 1×MES buffer. The positive clones weredesignated pTAP317 and subjected to sequence analysis. Thepolynucleotide sequence of MBP-zcytor19 fusion within pTAP317 is shownin SEQ ID NO:32, and the corresponding polypeptide sequence of theMBP-zcytor19 fusion is shown in SEQ ID NO:33.

B. Bacterial Expression of Human Zcytor19.

Ten microliters of sequencing DNA was digested with Not1 (NEB) in thefollowing reaction to remove the CEN-ARS: 10 μl DNA, 3 μl buffer3 (NEB),15 μl water, and 2 μl Not1 (10 U/μl NEB) at 37° C. for one hour. Then 7μl of the digest was mixed with 2 μl of 5× buffer and T4DNA ligase (1μ/μl BRL). Reaction was incubated at room temperature for one hour. Onemicroliter of the reaction was transformed into the E. coli strain W3110(ATCC). The cells were electropulsed at 2.0 kV, 25 μF and 400 ohms.Following electroporation, 0.6 ml SOC (2% Bacto™ Tryptone (Difco,Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added to the cells. After aone hour incubation at 37° C., the cells were plated in one aliquot onLB Kan plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 30 mg/LKanamycin). Individual clones were analyzed by diagnostic digests forthe absence of yeast marker and replication sequence.

A positive clone was used to inoculate an overnight starter culture ofSuperbroth II (Becton Dickinson) with 30 μg/ml of kanamycin. The starterculture was used to inoculate 4 2L-baffled flasks each filled with 500ml of Superbroth II+Kan. Cultures shook at 37° C. at 250 rpm until theOD₆₀₀ reached 4.1. At this point, the cultures were induced with 1mMIPTG. Cultures grew for two more hours at 37° C., 250 rpm at whichpoint 2 ml was saved for analysis and the rest was harvested viacentrifugation. Pellet was saved at −80° C. until transferred to proteinpurification.

Example 11 Purification Scheme for Zcytor19-FC4 Fusion

All procedures performed at 4C., unless otherwise noted. The conditionedmedia was concentrated first 20 times by using an Amicon/MilliporeSpiral cartridge, 10 kD MWCO. (at ambient temperature) The concentratedmedia was then applied to an appropriately sized POROS 50 A (coupledprotein A) column at an optimal capture flow rate. The column was washedwith 10 column volumes (CV) of equilibration buffer, then rapidly elutedwith 3 CV of 0.1 M Glycine pH 3. The collected fractions had apredetermined volume of 2M TRIS pH 8.0 added prior to the elution toneutralize the pH to about 7.2.

Brilliant Blue (Sigma) stained NuPAGE gels were ran to analyze theelution. Fractions of interested were pooled and concentrated using a 30kD MWCO centrifugal concentrator to a nominal volume. The concentratedProtein A pool was injected onto an appropriately sized PhamiciaSephacryl 200 column to remove aggregates and to buffer exchange theprotein into PBS pH 7.3.Brilliant Blue (Sigma) stained NuPAGE gels wereagain used to analyze the elution. Fractions were pooled. Western andBrilliant Blue (Sigma) stained NuPAGE gels were ran to confirm purityand content. For further analysis, the protein was submitted for AAA,and N-terminal sequencing. AAA analysis and N-terminal sequencingverified the zcytor19-Fc polyepptide; the N-terminal amino acid sequencewas as expected SRPRL APPQX VTLLS QNFSV (SEQ ID NO:34).

Example 12 Human Zcytor19 Expression Based on RT-PCR Analysis ofMultiple Tissue and Blood Fraction First-Strand cDNA Panels

Gene expression of zcytor19 was examined using commercially availablenormalized multiple tissue first-strand cDNA panels (OriGeneTechnologies, Inc. Rockville, Md.; BD Biosciences Clontech, Palo Alto,Calif.). These included OriGene's Human Tissue Rapid-Scan™ Panel(containing 24 different tissues) and the following BD BiosciencesClontech Multiple Tissue cDNA (MTC™) Panels: Human MTC Panel I(containing 8 different adult tissues), Human MTC Panel II (containing 8different adult tissues), Human Fetal MTC Panel (containing 8 differentfetal tissues), Human Tumor MTC Panel (containing carcinomas from 7different organs), Human Blood Fractions MTC Panel (containing 9different blood fractions), and Human Immune System MTC Panel(containing 6 different organs and peripheral blood leukocyte).

PCR reactions were set up using zcytor19 specific oligo primers ZC40285(SEQ ID NO:35) and ZC40286 (SEQ ID NO:36) which yield a 426 bp product,Qiagen HotStarTaq DNA Polymerase (Qiagen, Inc., Valencia, Calif.) andRediLoadm™ dye (Research Genetics, Inc., Huntsville, Ala.). The PCRcycler conditions were as follows: an initial 1 cycle 15 minutedenaturation at 95° C., 35 cycles of a 45 second denaturation at 95° C.,1 minute annealing at 63° C. and 1 minute and 15 seconds extension at72° C., followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 2% agarose gel (EMScience, Gibbstown, N.J.) and visualized by staining with ethidiumbromide.

A DNA fragment of the correct size was observed in the following humanadult tissues: adrenal gland, bone marrow, colon, heart, liver, lung,lymph node, muscle, ovary, pancreas, placenta, prostate, salivary gland,small intestine, spleen, stomach, testis, thyroid, and tonsil. A DNAfragment of the correct size was observed in the following human fetaltissues: heart, liver, lung, kidney, skeletal muscle, spleen, andthymus. A DNA fragment of the correct size was observed in the followinghuman blood fractions: peripheral blood leukocyte, mononuclear cells(B-cells, T-cells, and monocytes), resting CD8+ cells(T-suppressor/cytotoxic), resting CD19+ cells (B-cells), activated CD19+cells, activated mononuclear cells, and activated CD4+ cells. A DNAfragment of the correct size was observed in the following tumortissues: breast carcinoma, colon adenocarcinoma, lung carcinoma, ovariancarcinoma, pancreatic adenocarcinoma, and prostatic adenocarcinoma.

Because zcytor19 is expressed in these specific tumor tissues, zcytor19polynucleotides, polypeptides and antibodies can be used as a tumormarker as disclosed herein. Moreover, an antibody to zcytor19 could haveanti-tumor activity, as well as toxin-conjugates, cytokine conjugates orother conjugates of an antibody, or the zcytor19 receptor ligand itself.The antagonist of zcytor19 ligand, such as anti-zcytor19 antibodies orsoluble receptors can also act as anti-tumor reagents.

Example 13 Generation and Analysis of Zcytor19 KO Mice

A. Identification of BAC Clones Positive for Mouse Zcytor19 Gene

One BAC clone positive for mouse zcytor19 gene was identified usingIncyte Genomic's (St. Louis, Mo.) Easy-to-Screen DNA Pools, BAC Mouse ES(Release I) following Manufacturer's instructions. Oligonucleotides weredesigned to generate a PCR fragment containing partial exon 6, completeintron 6 and partial exon 7 sequences.

PCR reactions were carried out in 25 μl using 1.75 units of Advantage 2polymerase (Clontech). Either 2 μl or 10 μl of BAC library DNA was usedas template in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH₄)₂SO₄,6.7 mM MgCl₂.

5 mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% Dimethyl Sulfoxide, 1 mMdeoxynucleotides, 140 nM forward primer ZC39128 (SEQ ID NO:37) and 140nM reverse primer ZC39129 (SEQ ID NO:38). PCR conditions were as follows95° C. for 1 min,; 30 cycles of 95° C. for 15 seconds, 55° C. for 30seconds, and 68° C. for 30 seconds; and 68° C. for 2 minutes; followedby a 4° C. hold. PCR products were analyzed by agarose gelelectrophoresis. Positive PCR products were found to be 1,149 bp.

Four additional BAC clones positive for mouse zcytor19 gene wereidentified using Incyte's BAC Mouse Filter Set (Release II) followingManufacturer's instructions. Oligonucleotides were designed to generatea PCR fragment containing partial exon 6, and partial exon 7 sequencesfrom mouse cDNA template.

PCR reactions were carried out in 25 μl using 1.75 units of Advantage 2polymerase (Clontech). 2 μl of Neonatal Mouse skin cDNA library (JAK062700B) was used as template in buffer containing 67 mM Tris pH 8.8,16.6 mM (NH₄)₂SO₄, 6.7 mM MgCl₂.

5 mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% Dimethyl Sulfoxide, 1 mMdeoxynucleotides, 140 nM forward primer ZC39128 (SEQ ID NO:37) and 140nM reverse primer ZC39129 (SEQ ID NO:38). PCR conditions were asdescribed above. PCR products were separated by agarose gelelectrophoresis and purified using Qiaquick (Qiagen) gel extraction kit.The isolated, approximately 400 bp, DNA fragment was labeled usingPrime-It II (Stratagene) Random Primer labeling kit and purified usingMicroSpin S-200HR columns (AmershamPharmacia).

The labeled probe was used to screen Incyte's 7 filter BAC library set.Hybridizations were carried out at 55° C. overnight using ExpressHyb(Clontech). Filters were then washed 3 times for 30 minutes at 50° C.with 0.1×SSC, 01% SDS, autoradiographed overnight and compared tomanufacturer's grid patterns to identify positive clones.

B. Characterization of Zcytor19 Mouse Positive BACs.

Five zcytor19 mouse positive BAC clones from 129/SvJ Embryonic Stem Celllibraries (Release I and II) were obtained from Incyte Genomics. BACclones were grown within Escherichia coli host strain DH10B in liquidmedia and extracted using BAC large plasmid purification kit MKB-500(Incyte Genomics) according to manufacturer's instructions. 4 of 5 BACswere found to contain at least 2,000 bp of 5′ untranslated region,exon1, and exon 5 as determined by PCR. 100 ng of each BAC DNA was usedas template using the following conditions: PCR reactions were carriedout in 25 μl using 1.75 units of Advantage 2 polymerase (Clontech) inbuffer containing 67 mM Tris pH 8.8, 16.6 mM (NH₄)₂SO_(4,), 6.7 mMMgCl₂, 5 mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% DimethylSulfoxide, 1 mM deoxynucleotides, 140 nM forward and 140 nM reverseprimer. PCR conditions were as follows 95° C. for 1 min,; 30 cycles of95° C. for 15 seconds, 55° C. for 30 seconds, and 68° C. for 30 seconds;and 68° C. for 2 minutes; followed by a 4° C. hold. PCR products wereanalyzed by agarose gel electrophoresis. Using forward primer ZC40784(SEQ ID NO:39) and reverse primer ZC40785 (SEQ ID NO:40) partial 5′ UTRwas amplified and found to be 957 bp. Using forward primer ZC40786 (SEQID NO:41) and reverse primer ZC40787 (SEQ ID NO:42) partial 5′ UTR,complete exon 1 and partial intron 1 was amplified and found to beapproximately 950 bp. Using forward primer ZC39128 (SEQ ID NO:37) andforward primer ZC39129 (SEQ ID NO:38) containing partial exon 6,complete intron 6 and partial exon 7 sequence was amplified and found tobe 1,149 bp.

Four of the 5 BAC clones were found to contain at least 3,796 bp of 5′UTR and at 6,922 bp of 3′ UTR by Southern Blot analysis.Oligonucleotides ZC40784 (SEQ ID NO:39) and ZC39129 (SEQ ID NO:38) wereend labeled using T4 polynucleotide kinase (Roche) and used to probeSouthern Blots containing 5 BAC candidates digested with restrictionendonucleases EcoRI (Life Technologies) and XbaI (New England Biolabs).Results indicated 4 of 5 BACs contained at least 3,796 bp of 5′ UTR and5 of 5 BACs contained at least 6,922 bp of 3′ UTR.

C. Determination of Zcytor19 Mouse Intron 6 Sequence.

Oligonucleotides were designed to generate a PCR fragment containingpartial exon 6, complete intron 6 and partial exon 7 sequences.

PCR reactions were carried out in 25 μl using 1.75 units of Advantage 2polymerase (Clontech). 100 ng of 129/Sv mouse genomic DNA was used astemplate in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH₄)₂SO_(4,),6.7 mM MgCl₂, 5 mM 2-Mercaptoethanol, 100 μLg/ml gelatin, 10% DimethylSulfoxide, 1 mM deoxynucleotides, 140 nM forward primer ZC39128 (SEQ IDNO:37) and 140 nM reverse primer ZC39129 (SEQ ID NO:38). PCR conditionswere as described above. PCR products were analyzed by agarose gelelectrophoresis and found to be 1,149 bp. PCR products were thenpurified using Qiaquick (Qiagen) PCR purification kit. Determination ofintron 6 sequence was made by sequence analysis using oligos ZC39128(SEQ ID NO:37) and ZC 39129 (SEQ ID NO:38).

D. Determination of Zcytor19 Mouse Intron 5 Sequence

Oligonucleotides were designed to generate a PCR fragment containingpartial exon5, complete intron5 and partial exon6. PCR reactions werecarried out in 25 μl using 1.75 units of Advantage 2 polymerase(Clontech). 100 ng of 129/Sv mouse genomic DNA was used as template inbuffer containing 67 mM Tris pH 8.8, 16.6 mM (NH₄)₂SO_(4,), 6.7 mMMgCl_(2,), 5 mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% DimethylSulfoxide, 1 mM deoxynucleotides, 140 nM forward primer ZC39408 (SEQ IDNO:43) and 140 nM reverse primer ZC39409 (SEQ ID NO:44). PCR conditionswere as follows 95° C. for 1 min,; 30 cycles of 95° C. for 15 seconds,55° C. for 30 seconds, and 68° C. for 30 seconds; and 68° C. for 2minutes; followed by a 4° C. hold. PCR products were analyzed by agarosegel electrophoresis and found to be 356 bp. PCR products were thenpurified using Qiaquick (Qiagen) PCR purification kit. Determination ofintron 6 sequence was made by sequence analysis using oligos ZC39408(SEQ ID NO:43) and ZC 39409 (SEQ ID NO:44).

E. Design of Oligonucleotides for Generating of KO Constructs of theMouse Zcytor19 Gene

To investigate biological function of zcytor19 gene, a knockout mousemodel is being generated by homologous recombination technology inembryonic stem (ES) cells. In this model, the coding exon 1, 2 and 3 aredeleted to create a null mutation of the zcytor19 gene. This deletionremoves the translation initiation codon, the signal domain and part ofthe extracellular domain of the zcytor19 protein, thus inactivating thezcytor19 gene.

ET cloning technique will be used to generate the KO vector (Stewart etal, Nucl. Acids Res. 27:6, 1999) First, Kanomycin resistance cassette isused to replace intronsl, 2 and 3 of zcytor19 mouse gene. A forwardknockout oligonucleotide (SEQ ID NO:45) was designed to be 121nucleotides in length, having 52 bp of homology to the 5′ UTR ofzcytor19m a 42 bp linker having SfiI, FseI, BamHI and HindIIIrestriction sites and 27 bp of homology to the 5′ end of the Kanomycinresistance cassette. A reverse knockout oligonucleotide (SEQ ID NO:46)was designed to be 125 nucleotides in length, having 50 bp of homologyto intron 3 of zcytor19 mouse, a 48 bp linker having SfiI, AscI, BamHIand HindIII restriction sites and 27 bp of homology to the 3′ end of theKanomycin resistance cassette. The above oligonucleotides can be used tosynthesize a PCR fragment 1073 bp in length containing the entireKanomycin resistance cassette with the first 52 bp having homology tothe 5′ UTR of zcytor19 mouse and the last 50 bp having homology tointron 3.

The fragment will then be used to construct a Knockout vector through ETCloning, in which zcytor19 mouse positive BAC cell hosts are madecompetent through treatment with glycerol then transfected with theplasmid pBADalpha/beta/gamma(Amp). Resistance to chloramphenical andampicillin selects for transformed cell. Cells are then re-transformedwith the Kanomycin PCR fragment containing homology arms. The Beta andgamma recombination proteins of pBADalpha/beta/gamma(Amp) are induced bythe addition of arabinose to the growth media through the activation ofthe Red alpha gene. Recombinant BACs are selected for by resistance tokanomycin and ampicillin then screened by PCR. Once a recombinant BAC isidentified a fragment is subcloned containing at least 1,800 bp ofsequence upstream of kanomycin resistance cassette insertion and atleast 6,000 bp of sequence downstream into a pGEM7 derived vector. TheKanomycin resistance cassette is then replaced by standard ligationcloning with a IRES/LacZ/Neo-MCl cassette. The IRES is an internalribosome entry sequence derived from encephalomyocarditis virus. It isfused in-frame to the reporter lacZ gene, linked to a polyA signal.Downstream of the IRES/LacZ reporter gene, MCl promoter drives theexpression of a G418 resistance selectable marker Neo gene. Theselectable maker cassette contains termination codons in all threereading frames. Thus, the drug resistance gene Neo is used for selectionof homologous recombination events in embryonic stem (ES) cells.IRES/LacZ reporter gene will be used to monitor the expression of thereplaced gene after homologous recombination. Homologous recombinationof the knockout vector and the target locus in ES cells leads to thereplacement of a total 17,980 bp, including complete exons 1, 2 and 3,of the wild type locus with the IRES/LacZ/Neo-MCl cassette, which isabout 5,200 bp in length.

F. Generation of Zcytor19 KO Mice

The KO vector, described above, is linearized by PmeI digestion, andelectroporated into ES cells. Homologous recombination events areidentified by PCR screening strategy, and confirmed by Southern BlotAnalysis, using a standard KO protocol. See, A. L. Joyner, GeneTargeting. A Practical Approach. IRL Press 1993.

Once homologous recombination events are identified, ES cells will beexpanded, and injected into blastocysts to generate chimeras. Chimericmales will be used to breed to C57black females to achieve germ linetransmission of the null mutation, according to standard procedures. SeeHogan, B. et al., Manipulating the Mouse Embryo. A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1994.

Heterozygous KO animals will be bred to test biological functions of thezcytor19 gene. Of offspring produced, ¼ should be wild type, ½ should beheterozygous, and ¼ should be homozygous. Homozygous will be analyzed indetails as described below.

G. Microscopic Evaluation of Tissues from Zcytor19 Homozygous Animals.

Since zcytor19 is expressed in following tissues, we will examine thesetissues carefully: colon, ovary placenta, pituitary, lymph node, smallintestine, salivary gland, rectum, prostate, testis, brain, lung,kidney, thyroid, spinal cord, bone marrow, and cervix.

Spleen, thymus, and mesenteric lymph nodes are collected and preparedfor histologic examination from transgenic animals expressing zcytor19.Other tissues which are routinely harvested included the following:Liver, heart, lung, spleen, thymus, mesenteric lymph nodes, kidney,skin, mammary gland, pancreas, stomach, small and large intestine,brain, salivary gland, trachea, esophagus, adrenal, pituitary,reproductive tract, accessory male sex glands, skeletal muscle includingperipheral nerve, and femur with bone marrow. The tissues are harvestedfrom homozygous animals as well as wild type controls. Samples are fixedin 10% buffered formalin, routinely processed, embedded in paraffin,sectioned at 5 microns, and stained with hematoxylin and eosin. Theslides are examined for histological, and pathological changes, such asinflammatory reactions, and hypo-proliferation of certain cell types.

H. Flow Cytometric Analysis of Tissues from Homozygous Mouse MutantsMissing Zcytor19.

Homozygous animals missing zcytor19 gene are to be sacrificed for flowcytometric analysis of peripheral blood, thymus, lymph node, bonemarrow, and spleen.

Cell suspensions are made from spleen, thymus and lymph nodes by teasingthe organ apart with forceps in ice cold culture media (500 ml RPMI 1640Medium (JRH Biosciences. Lenexa, KS); 5 ml 100× L-glutamine (Gibco BRL.Grand Island, N.Y.); 5 ml 100× Na Pyruvate (Gibco BRL); 5 ml 100×Penicillin, Streptomycin, Neomycin (PSN) (Gibco BRL) and then gentlypressing the cells through a cell strainer (Falcon, VWR Seattle, Wash.).Peripheral blood (200 ml) is collected in heparinized tubes and dilutedto 10 mls with HBSS containing 10 U Heparin/ml. Erythrocytes are removedfrom spleen and peripheral blood preparations by hypotonic lysis. Bonemarrow cell suspensions are made by flushing marrow from femurs withice-cold culture media. Cells are counted and tested for viability usingTrypan Blue (GIBCO BRL, Gaithersburg, Md.). Cells are resuspended in icecold staining media (HBSS, 1% fetal bovine serum, 0.1% sodium azide) ata concentration of ten million per milliliter. Blocking of Fc receptorand non-specific binding of antibodies to the cells was achieved byadding 10% normal goat sera and Fc Block (PharMingen, La Jolla, Calif.)to the cell suspension.

Cell suspensions are mixed with equal volumes of fluorochrome labeledmonoclonal antibodies (PharMingen), incubated on ice for 60 minutes andthen washed twice with ice cold wash buffer (PBS, 1% fetal bovine serum,0.1% sodium azide) prior to resuspending in 400 ml wash buffercontaining lmg/ml 7-AAD (Molecular Probes, Eugene, Oreg.) as a viabilitymarker in some samples. Flow data was acquired on a FACSCalibur flowcytometer (BD Immunocytometry Systems, San Jose, Calif.). Bothacquisition and analysis were performed using CellQuest software (BDImmunocytometry Systems).

The cell populations in all lymphoid organs will be analyzed to detectabnormalities in specific lineages of T cell, B cell, or otherlymphocytes, and cellularity in these organs.

Example 14 Identification of Cells Expressing Zcytor19 Using In SituHybridization

Human tissues from cervical carcinoma, normal and carcinoma colon,duodenum, endometrial carcinoma, normal and carcinoma ovary, uterus,heart, liver, lung, muscle sarcoma, and normal and carcinoma skin werescreened for zcytor19 expression by in situ hybridization. The tissueswere fixed in 10% buffered formalin and blocked in paraffin usingstandard techniques. Tissues were sectioned at 5 microns. Tissues wereprepared using a standard protocol. Briefly, tissue sections weredeparaffinized with HistoClear (National Diagnostics, Atlanta, Ga.) andthen dehydrated with ethanol. Next they were digested with Proteinase K(50 μg/ml) (Boehringer Diagnostics, Indianapolis, Ind.) at 23° C. for4-15 minutes. This step was followed by acetylation and re-hydration ofthe tissues.

One in situ probe was designed against the human zcytor19 sequence.Plasmid DNA 100933 was digested with restriction enzyme HindIII, whichcovers 0.7 kb from the end of 3′ UTR. The T-7 RNA polymerase was used togenerate an antisense probe. The probe was labeled with digoxigenin(Boehringer) using an In Vitro transcription System (Promega, Madison,Wis.) as per manufacturer's instruction.

In situ hybridization was performed with a digoxigenin- orbiotin-labeled zcytor19 probe (above). The probe was added to the slidesat a concentration of 1 to 5 pmol/ml for 12 to 16 hours at 60° C. Slideswere subsequently washed in 2×SSC and 0.1×SSC at 55° C. The signals wereamplified using tyramide signal amplification (TSA) (TSA, in situindirect kit; NEN) and visualized with Vector Red substrate kit (VectorLab) as per manufacturer's instructions. The slides were thencounter-stained with hematoxylin (Vector Laboratories, Burlingame,Calif.).

Positive signal were observed in most of carcinoma samples. In cervicalcarcinoma, carcinoma epithelial cells were positive. There were alsosome signals in a subset of lymphocytes in the lymphoid follicles.Similarly, both carcinoma and some immune cells were positive in thecolon carcinoma samples, while normal colon samples were negative. Weakstaining was also in the endometrial carcinoma and ovarian carcinoma,while normal ovary and uterus were negative. There was weak staining inthe cancer area of the muscle sarcoma sample. Keratinocytes werepositive in the skin carcinoma and Kaposi's sarcoma samples, while nostaining was observed in the normal skin. In heart and liver, a subsetof cells possibly circulating WBC, were positive for zcytor19. Itappears endothelial cells in some vessels may also be positive. In lung,type II pneumocytes and macrophage-like cells were positive. Bronchialepithelium and endothelium were also positive in some lung specimens. Insummary, zcytor19 appears to be up-regulated in carcinoma cells. Thereis low level of zcytor19 mRNA in a subset of lymphocytes and endothelialcells.

Because zcytor19 is expressed in these specific tumor tissues, zcytor19polynucleotides, polypeptides and antibodies can be used as a tumormarker as disclosed herein. Moreover, an antibody to zcytor19 could haveanti-tumor activity, as well as toxin-conjugates, cytokine conjugates orother conjugates of an antibody, or the zcytor19 receptor ligand itself.The antagonist of zcytor19 ligand, such as anti-zcytor19 antibodies orsoluble receptors can also act as anti-tumor reagents.

Example 15 Construction of BaF3 Cells Expressing the Zcytor19 Receptor(BaF3 Zcytor19 cells) with Puromycin Resistant and Zeomycin ResistantVectors

Two types of BaF3 cells expressing the full-length zcytor19 receptorwere constructed using 30 μg of zcytor19 expression vectors, oneresistant to puromycin, one resistant to zeomycin described below. TheBaF3 cells expressing the zcytorl 9 receptor mRNA with puromycinresistance were designated as BaF3/zcytor19-p. The BaF3 cells expressingthe zcytor19 receptor mRNA with zeomycin resistance were designated asBaF3/zcytor19-z.

A. Construction of BaF3 Cells Expressing the Zcytor19 Receptor

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2mM L-glutaMax-1™(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL). Prior to electroporation,pZP-5N/CRF2-4 was prepared and purified using a Qiagen Maxi Prep kit(Qiagen) as per manufacturer's instructions. BaF3 cells forelectroporation were washed twice in PBS (Gibco BRL) and thenresuspended in RPMI media at a cell density of 10⁷ cells/ml. One ml ofresuspended BaF3 cells was mixed with 30 μg of the pZP-7p/zcytor19plasmid DNA, or 30 μg of the pZP-7z/zcytor19 plasmid DNA, andtransferred to separate disposable electroporation chambers (GIBCO BRL).The cells were given two serial shocks (800 1 Fad/300 V.; 1180 1Fad/300V.) delivered by an electroporation apparatus (CELL-PORATOR™M; GIBCOBRL), with a 1 minute rest between the shocks. After a 5 minute recoverytime, the electroporated cells were transferred to 50 ml of completemedia and placed in an incubator for 15-24 hours (37° C., 5% CO₂). Thecells were then spun down and resuspended in 50 ml of complete mediacontaining Puromycin (Clonetech) selection (2 μg/ml) for the cellstransfected with pZP-7p/zcytor19, or Zeocin selection (1:150-1:333) forthe cells transfected with pZP-7p/zcytor19, and placed in a T-162 flaskto isolate the antibiotic-resistant pools. Pools of the transfected BaF3cells, hereinafter called BaF3/zcytor19-puro and BaF3/zcytor19-zeocells, were assayed for expression of zcytor19 by RT-PCR.

B. Confirmation of Zcytor19 Expression by RT-PCR.

The BaF3/zcytor19-puro and BaF3/zcytor19-zeo cells were harvested forRNA, which was then put into a reverse transcriptase reaction, andsubsequently tested by PCR for the presence of zcytor19.

Flasks of cells were grown to confluence, then 10 ml were removed andspun down to obtain a cell pellet. RNA was purified from the pelletusing the RNeasy Total RNA Purification kit, with the additionalRNase-free DNase set (Qiagen), following the manufacturer's protocol.Reverse transcription was then done on the samples using theStrataScript RT-PCR kit (Stratagene), following the manufacturer'sprotocol through the completion of the RT reaction. PCR was then done bymixing 0.2 pmol each of primers ZC40279 and ZC37863, 0.2 mM of dNTP mix(Roche) containing equal amounts of each nucleotide, 5 μl of 10× cDNAPCR Reaction Buffer (Clonetech), 3μl DNA from the RT reaction, 0.5 μlAdvantage2 Polymerase (Clonetech), made to a final volume of 50 μl withwater. The reaction ran for 95° C., 5 min, then 30 cycles of 95° C. 30sec, 60° C. 30 sec, 72° C. 1 min, then 72° C. 7 min and a 4° C. soak, ona Perkin Elmer GeneAmp PCR System 2400. The samples were mixed with 3 mlloading dye, and 25 ml was run on a 1% OmniPur Agarose (Merck) gel.Zcytor19 bands were detected on the gel for both BaF3/zcytor19-puro andBaF3/zcytor19-zeo, indicating that those cells are expressing the gene.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for detecting rectal tumor in a patient, comprising:obtaining rectal tissue from the patient; incubating the tissue with anantibody that specifically binds to the polypeptide encoded by thepolynucleotide consisting of the nucleic acid sequence as shown in SEQID NO: 18 from nucleotide 238 to nucleotide 496 under conditions whereinthe antibody binds to its complementary polypeptide the in the tissue;and detecting the antibody bound in the tissue, and wherein an increasein the amount of antibody bound in the tissue compared to the amount ofantibody bound to a normal rectal control tissue indicates rectal tumor.2. The method according to claim 1, wherein the antibody is a purifiedmixture of polyclonal antibodies.
 3. The method according to claim 1,wherein the antibody is a monoclonal antibody.
 4. A method for detectingrectal tumor in a patient, comprising: obtaining rectal tissue from thepatient; incubating the tissue with an antibody that specifically bindsto the polypeptide selected from the group consisting of: (a) thepolypeptide consisting of the amino acid sequence as shown in SEQ ID NO:19from amino acid 21 to amino acid 223; and (b) the polypeptideconsisting of the amino acid sequence as shown in SEQ ID NO: 19fromamino acid 21 to amino acid 226; under conditions wherein the antibodybinds to its complementary polypeptide in the tissue; and detecting theantibody bound in the tissue, and wherein an increase in the amount ofantibody bound in the tissue compared to the amount of antibody bound toa normal rectal control tissue indicates rectal tumor.
 5. The methodaccording to claim 4, wherein the antibody is a purified mixture ofpolyclonal antibodies.
 6. The method according to claim 4, wherein theantibody is a monoclonal antibody.
 7. A method for detecting skin cancerin a patient, comprising: obtaining skin tissue from the patient;incubating the tissue with an antibody that specifically binds to thepolypeptide selected from the group consisting of: (a) the polypeptideconsisting of the amino acid sequence as shown in SEQ ID NO: 19fromamino acid 21 to amino acid 520; (b) the polypeptide consisting of theamino acid sequence as shown in SEQ ID NO: 19from amino acid 1 to aminoacid 520; and (c) the polypeptide consisting of the amino acid sequenceas shown in SEQ ID NO: 19from amino acid 250 to amino acid 520; underthe conditions wherein the antibody binds to its complementarypolypeptide in the tissue; and detecting the antibody bound in thetissue, and wherein an increase in the amount of antibody bound in thetissue compared to the amount of antibody bound to a normal skin controltissue indicates skin cancer.
 8. The method according to claim 7,wherein the antibody is a purified mixture of polyclonal antibodies. 9.The method according to claim 7, wherein the antibody is a monoclonalantibody.